Vaccine compositions and methods of use thereof

ABSTRACT

Nanoparticle-based vaccines, compositions, kits and methods are used for the effective delivery of one or more antigens in vivo for vaccination and antibody (e.g., monoclonal antibody) production, and for the effective delivery of peptides, proteins, siRNA, RNA or DNA to PAPCs or MHC class II positive cells (e.g. tumor cells). Antigens may be, for example, DNA that results in expression of the gene of interest and induction of a robust and specific immune response to the expressed protein in a subject (e.g., mammal). Antigens may also be immunogenic peptides or polypeptides that are processed and presented. In one embodiment, a nanoparticle-based method to deliver antigens in vivo as described herein includes injection of a vaccine composed of a DNA encoding at least one antigen, or at least one antigenic peptide or polypeptide conjugated to a charged dendrimer (e.g., PADRE-derivatized dendrimer) that is also conjugated to a T helper epitope (e.g., PADRE). Negatively-charged plasmids bind naturally to a positively-charged PADRE-dendrimer, while peptide or polypeptide antigens can be chemically linked to the PADRE-dendrimer if they are not negatively-charged. Alternatively, negatively-charged dendrimers may be used. The compositions, kits, vaccines and methods described herein have both prophylactic and treatment applications, i.e., can be used as a prophylactic to prevent onset of a disease or condition in a subject, as well as to treat a subject having a disease or condition. A vaccine as described herein can be used to mount an immune response against any infectious pathogen or cancer.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.13/262,285, filed on Dec. 19, 2011, which is a § 371 national phaseentry of International application number PCT/US2010/29694, filed Apr.1, 2010, which claims priority to U.S. provisional patent applicationNo. 61/165,732 filed Apr. 1, 2009, the contents of which are herebyincorporated herein by reference in their entireties.

REFERENCE TO A SEQUENCE LISTING

This application includes a “Sequence Listing” which is provided as theelectronic file “7230612CON-Sequence.txt” (9,640 bytes) created on Sep.15, 2017, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates generally to the fields of chemistry andimmunology. More particularly, the invention relates to vaccines,compositions and methods for inducing an immune response in a subject.

BACKGROUND

The prevention of microbial infections and pathogenic processes via theuse of vaccines is considered one of the most effective and desirableprocedures to combat illness. Antigens or immunogens are introduced intoan organism in a manner that stimulates an immune response in the hostorganism in advance of an infection or disease. Traditional vaccinestrategies, however, have not been effective in mounting protectionagainst many pathogens or cancers. Of more than 100 pathogens, onlyabout 20 successful vaccines have been made by traditional vaccinestrategies. Of those vaccines that induce a high cytotoxic T lymphocyte(CTL) response, some often show a modest objective response rate due topoor immunogenicity, immuno-avoidance mechanisms, and deceptiveimprinting. Current methods of vaccine delivery have a modest successrate in terms of inducing protective immune responses because they donot induce robust “danger signals,” they initiate inhibitory responsesthat act as feedback mechanisms, and they deliver antigens tononprofessional antigen presenting cells (APCs). Current cancervaccines, for example, even when mounting a high CTL response, show amodest (2.6%) objective response rate. They are associated with a numberof disadvantages, including poor immunogenicity and immuno-avoidancemechanisms. Moreover, the most promising cancer vaccines (dendriticcell-based and G-vax-based), are extremely costly and preparation ofthese vaccines is very involved (e.g., requiring personalization and GMPmanufacturing).

Genetic vaccination or genetic immunization, which involves theinoculation of genetic materials into mammalian hosts to produceantigens, is considered a possible approach for vaccines includingcancer vaccines. The delivered mammalian expression vector encoding theantigen of interest results in in vivo expression and subsequently tothe development of antigen-specific responses. In addition, genes arenegatively-charged polymers, which cannot cross cell membranes and reachthe cell nucleus, where they can express a protein of interest. Geneticvaccination offers a number of advantages, including generation of afull spectrum of native epitopes expressed in vivo, achievement of thenative conformation of a protein compared with administration ofrecombinant protein expressed in vitro, induction of antibody andcellular immune responses, and elimination of the need for costly andcommonly challenging steps for antigen production. Genetic vaccination,however, is associated with a number of disadvantages including breakingtolerance to self antigens, poor in vivo delivery of nucleic acids intothe cell and nucleus, a lack of specificity for particular types ofcells, and weak immune responses.

Other forms of vaccines are associated with drawbacks as well. Forexample, viral delivery of genes results in strong immune responses toviral vectors and is associated with safety concerns. Proteinpurification from bacteria and production of peptides for use asantigens is expensive and time consuming.

Currently, there are no cost-effective, efficacious forms of vaccinesthat target APCs to produce specific and robust immune responses with noor few side effects. There is thus a significant need for a vaccine thattargets professional APCs and elicits a strong and specific cellular andantibody response and that is safe, cost-effective and easy to use.

SUMMARY

Described herein are nanoparticle-based compositions, kits and methodsand platforms for delivering an antigen or a nucleic acid encoding anantigen to professional APCs (PAPCs) in vivo that result in a robust andspecific immune response to the antigen. Also described herein arenanoparticle-based compositions, kits, methods and platforms fordelivering siRNA to PAPCs, and for delivering nucleic acids, peptides orproteins to cells (e.g., MHC Class II expressing tumor cells). A majordeficit of current vaccine strategies is that they induce suppressorcells including regulatory T cells. Targeted delivery of antigen toPAPCs is known to reduce or inhibit the activation of suppressormechanisms, in particular, those of regulatory T cells. The composition,kits and methods involve the combined use of MHC targeting andimmunogenic peptides (e.g., PADRE, HA) with charged (e.g.,positively-charged) highly branched polymeric dendrimers (e.g., PAMAMand other dendrimers) as vehicles for the targeted delivery of nucleicacids, peptides or polypeptides to specific cells, giving rise to a newnanoparticle-based method for genetic or protein vaccination. Typicalvaccines described herein include a charged (e.g., positively-charged)highly branched polymeric dendrimer conjugated to an MHC targeting andimmunogenic peptide such as T helper peptide (e.g., an epitope such asthe PADRE peptide or Influenza HA), at least one polypeptide antigen ora nucleic acid encoding the at least one antigen, and optionally PolyI-C. The positively-charged highly branched polymeric dendrimersdescribed herein effectively bind negatively-charged biomoleculesincluding DNA, RNA and others. Charged (e.g., positively-charged) highlybranched polymeric dendrimers conjugated to a T helper peptide (e.g., anepitope such as the PADRE peptide or Influenza HA) provide vaccines withincreased efficacy due to specific antigen delivery to PAPCs. In theexperiments described herein, the first use of PADRE to target PAPCs viaits binding to MHC class II molecules is shown. The experimentsdescribed herein describe effective use of two different targetingpeptides, whose unique feature is to bind to the MHC class II. Thus, thevaccines, methods and compositions described herein encompass all MHCclass II binding peptides. The vaccines, kits and compositions describedherein provide for specific and efficient transfection of PAPCs in vivo,and built-in universal T helper activity universally that result inmaturation of autologous PAPCs and hence robust and specific immuneresponses.

Dendrimers are an ideal DNA delivery candidate for they providestructural control over size and shape (cargo-space), are biocompatible(non-toxic and nonimmunogenic), have precise scaffolding properties,have a well-defined surface-modifiable functionality for specifictargeting moieties, have the ability for cellular adhesion andendocytosis and delivery into the cytoplasm or nucleus, have acceptablebiodegradation (the ability to safely degrade within the body), and areassociated with easy and consistently reproducible (clinical grade)synthesis. In the experiments described herein, the DNA, siRNA, peptideor polypeptide-conjugated positively-charged highly branched polymericdendrimer includes a peptide (e.g., PADRE or Influenza HA) that targetsAPCs and activates helper T cells in both humans and mice. The PADREpeptide has 2 main functions: escorting DNA to PAPCs as it binds to theMHC class-II present on the PAPCs and it stimulates T helper cells thatpromote the generation of cytotoxic T cells and the class switchingrequired for antibody responses. This novel nanoconstruct has uniqueproperties for gene and peptide delivery and vaccination. Theexperiments described herein also show that positively-charged highlybranched polymeric dendrimers (PAMAM dendrimers) conjugated to PADREdelayed the growth of and reduced the size of established and highlyaggressive B16/LU8 melanoma tumors in C57BL mice by 50% in a therapeuticsetting and demonstrated 100% eradication of tumors in a B16/OVApreventative setting, induced robust immune responses against a geneproduct used for vaccination, demonstrated transfection efficiency inboth mouse and human APCs by 2- or 3-fold, delivered a plasmid encodingGFP in vivo resulting in draining lymph nodes, and efficiently deliveredsiRNA into human B cells, T cells, and murine macrophages.

The compositions and vaccines described herein are a tailored and idealplatform for vaccination, as they target MHC class II positive cells,all or nearly all of which are PAPCs. However, as importantly, MHC classII positive cells express very important co-inhibitory andco-stimulatory molecules (including but not limited to CD80, CD86,B7-H1, B7-H4, B7-DC, CD137, OX40, Foxp3 and their putativeco-stimulatory receptor(s)) which suppress or promote T-cell activation.Targeted manipulation of the expression of molecules involved in thesepathways can be used for i) the immunotherapy/vaccination for cancer,infectious diseases or other novel vaccine approaches such asvaccination for addiction or infertility or neutralizing adisease-inducing agent in a subject, as well as management ofautoimmunity. Targeted delivery of vaccines to APCs as described hereinoffers a solution to the challenges associated with current vaccinationstrategies by resulting in much more robust immune responses, areduction of suppressor/feedback mechanisms, and preventing toxicity bylowering the vaccine dose.

Accordingly, described herein is a vaccine including at least onecharged highly branched polymeric dendrimer having conjugated thereto atleast one T helper peptide and a nucleic acid encoding at least oneantigen, wherein the at least one T helper peptide and the nucleic acidare conjugated to the exterior surface of the at least one chargedhighly branched polymeric dendrimer such that the at least one T helperpeptide specifically binds to professional antigen presenting cells andthe combination of the at least one T helper peptide, at least onecharged highly branched polymeric dendrimer, and nucleic acid are ableto induce an immune response against the at least one antigen. In thevaccine, the at least one dendrimer can be bound toPolyinosinic-polycytidylic acid. This embodiment can include apharmaceutically acceptable carrier and/or a water-in-oil emulsion. Inone embodiment, the at least one T helper peptide is a Pan-DR epitope(PADRE), e.g., two PADRE epitopes each having the amino acid sequence ofSEQ ID NO:1. The at least one T helper peptide can also be influenza HA.The nucleic acid can be an expression vector and the at least oneantigen can be a cancer antigen or an antigen from an infectiouspathogen. The at least one charged highly branched polymeric dendrimercan be a PAMAM dendrimer.

Also described herein is a vaccine including at least one charged highlybranched polymeric dendrimer having conjugated thereto at least one Thelper peptide and at least one peptide or polypeptide antigen, whereinthe at least one T helper peptide and the at least one peptide orpolypeptide antigen are conjugated to the exterior surface of the atleast one charged highly branched polymeric dendrimer such that the atleast one T helper peptide specifically binds to professional antigenpresenting cells and the combination of the at least one T helperpeptide, at least one charged highly branched polymeric dendrimer and atleast one peptide or polypeptide antigen are able to induce an immuneresponse against the at least one peptide or polypeptide antigen. In oneembodiment, the at least one charged highly branched polymeric dendrimerhas further conjugated thereto a second peptide or polypeptide antigenthat is different from the at least one peptide or polypeptide antigen.The vaccine can further include a second charged highly branchedpolymeric dendrimer having conjugated thereto at least one T helperpeptide and a second peptide or polypeptide antigen that is differentfrom the at least one peptide or polypeptide antigen, wherein the atleast one T helper peptide and the second peptide or polypeptide antigenare conjugated to the exterior surface of the second charged highlybranched polymeric dendrimer such that the at least one T helper peptidespecifically binds to professional antigen presenting cells and thecombination of the at least one T helper peptide, the second chargedhighly branched polymeric dendrimer and the second peptide orpolypeptide antigen are able to induce an immune response against thesecond peptide or polypeptide antigen. The at least one charged highlybranched polymeric dendrimer can be bound to Polyinosinic-polycytidylicacid. The vaccine can further include a pharmaceutically acceptablecarrier and/or a water-in-oil emulsion. The at least one T helperpeptide can be a Pan-DR epitope, e.g., two Pan-DR epitopes each havingthe amino acid sequence of SEQ ID NO:1. In another embodiment, the atleast one T helper epitope is influenza HA. The at least one peptide orpolypeptide antigen can be a cancer antigen or an antigen from aninfectious pathogen. The at least one charged highly branched polymericdendrimer can be a PAMAM dendrimer.

Further described herein is a method of delivering an antigen to amammal and inducing production of monoclonal antibodies against theantigen in the mammal. The method includes the steps of: administeringto the mammal a composition including at least one charged highlybranched polymeric dendrimer having conjugated thereto at least one Thelper peptide and at least one peptide or polypeptide antigen or anucleic acid encoding the at least one antigen, wherein the at least oneT helper peptide and the nucleic acid or at least one peptide orpolypeptide antigen are conjugated to the exterior surface of the atleast one charged highly branched polymeric dendrimer such that the atleast one T helper peptide specifically binds to professional antigenpresenting cells and the combination of the at least one T helperpeptide, at least one charged highly branched polymeric dendrimer, andthe nucleic acid or at least one peptide or polypeptide antigen are ableto induce an immune response against the at least one peptide orpolypeptide antigen, the composition in an amount effective to induceMHC class II mediated activation of helper T cells, whereinadministering the composition to the mammal results in production ofmonoclonal antibodies against the at least one peptide or polypeptideantigen. In an embodiment wherein the mammal has cancer, the at leastone peptide or polypeptide antigen is a cancer antigen, and thecomposition is a vaccine for the cancer. Typically, administration ofthe composition results in no local adverse reactions in the mammal. Inanother embodiment wherein the mammal has an infectious disease, the atleast one peptide or polypeptide antigen is from an infectious pathogen,and the composition is a vaccine for the infectious pathogen, typicallyresulting in no local adverse reactions in the mammal. The at least onecharged highly branched polymeric dendrimer can be bound toPolyinosinic-polycytidylic acid and/or can include a pharmaceuticallyacceptable carrier and/or water-in-oil emulsion.

In one embodiment of this method, the at least one T helper peptide is aPADRE epitope, e.g., two PADRE epitopes each having the amino acidsequence of SEQ ID NO:1. The at least one T helper peptide can also beinfluenza HA. The at least one charged highly branched polymericdendrimer can be a PAMAM dendrimer. The at least one charged highlybranched polymeric dendrimer can be further conjugated to a secondpeptide or polypeptide antigen that is different from the at least onepeptide or polypeptide antigen. The composition can further include asecond charged highly branched polymeric dendrimer having conjugatedthereto at least one T helper peptide and a second peptide orpolypeptide antigen that is different from the at least one peptide orpolypeptide antigen, wherein the at least one T helper peptide and thesecond peptide or polypeptide antigen are conjugated to the exteriorsurface of the second charged highly branched polymeric dendrimer suchthat the at least one T helper peptide specifically binds toprofessional antigen presenting cells and the combination of the atleast one T helper peptide, the second charged highly branched polymericdendrimer and the second peptide or polypeptide antigen are able toinduce an immune response against the second peptide or polypeptideantigen.

In one embodiment for producing and harvesting antibodies, the mammalcan be a rodent or rabbit and the monoclonal antibodies are harvestedfrom the mammal. In this embodiment, the monoclonal antibodies areprepared by the steps of: harvesting the antibodies from the mammal,titering the antibodies, removing the spleen from the mammal, andperforming fusion with myeloma. The antibodies can be humanized.

Further described herein is a composition including at least one chargedhighly branched polymeric dendrimer having conjugated thereto at leastone T helper peptide and at least one siRNA, wherein the at least one Thelper peptide and the at least one siRNA are conjugated to the exteriorsurface of the charged highly branched polymeric dendrimer such that theat least one T helper peptide specifically binds to professional antigenpresenting cells. The at least one charged highly branched polymericdendrimer can be a PAMAM dendrimer, the at least one T helper peptidecan be PADRE, and the siRNA can be directed against, for example,CTLA-4, Foxp3, CD28, IDO or Arginase 1.

Yet further described herein is a method of delivering siRNA intoprofessional antigen presenting cells including the steps of: providinga composition including at least one charged highly branched polymericdendrimer having conjugated thereto at least one T helper peptide and atleast one siRNA, wherein the at least one T helper peptide and the atleast one siRNA are conjugated to the exterior surface of the chargedhighly branched polymeric dendrimer such that the at least one T helperpeptide specifically binds to professional antigen presenting cells; andadministering the composition to a mammalian subject under conditions inwhich the at least one charged highly branched polymeric dendrimerhaving conjugated thereto at least one T helper peptide and at least onesiRNA binds to a professional antigen presenting cell and the siRNAenters the professional antigen presenting cell. The charged highlybranched polymeric dendrimer can be a PAMAM dendrimer, the at least oneT helper peptide can be, for example, a PADRE, and the siRNA can bedirected against, for example, CTLA-4, Foxp3, CD28, IDO or Arginase 1.In one embodiment, the siRNA prevents expression of CTLA-4, Foxp3, CD28,IDO or Arginase 1 in the professional antigen presenting cell.

Still further described herein is a method of inhibiting proliferationof MHC Class II tumor cells (e.g., lymphoma or a portion of a lymphoma)or inducing apoptosis of MHC Class II tumor cells in a mammal. Thismethod includes the steps of: administering to the mammal a compositionincluding at least one positively-charged highly branched polymericdendrimer having conjugated thereto at least one T helper peptide andbound by a nucleic acid encoding a protein, wherein the at least one Thelper peptide and the nucleic acid are conjugated and bound to theexterior surface of the at least one positively-charged highly branchedpolymeric dendrimer such that the at least one T helper peptidespecifically binds to MHC Class II tumor cells and the combination ofthe at least one T helper peptide, at least one positively-chargedhighly branched polymeric dendrimer, and the nucleic acid or proteinencoded by the nucleic acid inhibit proliferation of MHC Class II tumorcells or induce apoptosis of MHC Class II tumor cells. Thepositively-charged highly branched polymeric dendrimer can be, forexample, a PAMAM dendrimer and the at least one T helper peptide can be,for example, a PADRE. However, any suitable positively-charged highlybranched dendrimers and T helper peptides can be used.

A method for delivering a nucleic acid to a cell as described hereinincludes contacting the cell with a composition including at least onepositively-charged highly branched polymeric dendrimer having conjugatedthereto at least one T helper epitope and at least one nucleic acidencoding a peptide or protein, wherein the at least one T helper epitopeand the nucleic acid are conjugated to the exterior surface of the atleast one positively-charged highly branched polymeric dendrimer suchthat the at least one T helper epitope specifically binds to the cell,and the combination of the at least one T helper epitope, at least onepositively-charged highly branched polymeric dendrimer, and the nucleicacid are internalized by the cell. In this method, the peptide orprotein is typically expressed within the cell. Although any suitablepositively-charged highly branched dendrimers and T helper peptides canbe used, the positively-charged highly branched polymeric dendrimer canbe a PAMAM dendrimer, for example, and the at least one T helper peptidecan be a PADRE, for example.

A composition for delivering a nucleic acid to a cell as describedherein includes at least one positively-charged highly branchedpolymeric dendrimer having conjugated thereto at least one T helperpeptide and at least one nucleic acid encoding a peptide or protein,wherein the at least one T helper peptide and the nucleic acid areconjugated to the exterior surface of the at least onepositively-charged highly branched polymeric dendrimer such that the atleast one T helper peptide specifically binds to the cell, and thecombination of the at least one T helper peptide, at least onepositively-charged highly branched polymeric dendrimer, and the nucleicacid are internalized by the cell. The positively-charged highlybranched polymeric dendrimer can be a PAMAM dendrimer, and the at leastone T helper peptide can be a PADRE, for example. However, any suitablepositively-charged highly branched dendrimers and T helper peptides canbe used.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs.

As used herein, a “nucleic acid” or a “nucleic acid molecule” means achain of two or more nucleotides such as RNA (ribonucleic acid) and DNA(deoxyribonucleic acid), and chemically-modified nucleotides. A“purified” nucleic acid molecule is one that is substantially separatedfrom other nucleic acid sequences in a cell or organism in which thenucleic acid naturally occurs (e.g., 30, 40, 50, 60, 70, 80, 90, 95, 96,97, 98, 99, 100% free of contaminants). The terms include, e.g., arecombinant nucleic acid molecule incorporated into a vector, a plasmid,a virus, or a genome of a prokaryote or eukaryote. Examples of purifiednucleic acids include cDNAs, fragments of genomic nucleic acids, nucleicacids produced polymerase chain reaction (PCR), nucleic acids formed byrestriction enzyme treatment of genomic nucleic acids, recombinantnucleic acids, and chemically synthesized nucleic acid molecules. A“recombinant” nucleic acid molecule is one made by an artificialcombination of two otherwise separated segments of sequence, e.g., bychemical synthesis or by the manipulation of isolated segments ofnucleic acids by genetic engineering techniques.

When referring to an amino acid residue in a peptide, oligopeptide orprotein, the terms “amino acid residue”, “amino acid” and “residue” areused interchangably and, as used herein, mean an amino acid or aminoacid mimetic joined covalently to at least one other amino acid or aminoacid mimetic through an amide bond or amide bond mimetic.

As used herein, “protein” and “polypeptide” are used synonymously tomean any peptide-linked chain of amino acids, regardless of length orpost-translational modification, e.g., glycosylation or phosphorylation.

When referring to a nucleic acid molecule, polypeptide, or infectiouspathogen, the term “native” refers to a naturally-occurring (e.g., awild-type (WT)) nucleic acid, polypeptide, or infectious pathogen.

As used herein, the term “antigen” or “immunogen” means a molecule thatis specifically recognized and bound by an antibody.

When referring to an epitope (e.g., T helper epitope), by biologicalactivity is meant the ability to bind an appropriate MHC molecule and,in the case of peptides useful for stimulating CTL responses, induce a Thelper response and a CTL response against a target antigen or antigenmimetic.

The terms “specific binding” and “specifically binds” refer to thatbinding which occurs between such paired species as enzyme/substrate,receptor/agonist, antibody/antigen, etc., and which may be mediated bycovalent or non-covalent interactions or a combination of covalent andnon-covalent interactions. When the interaction of the two speciesproduces a non-covalently bound complex, the binding which occurs istypically electrostatic, hydrogen-bonding, or the result of lipophilicinteractions. Accordingly, “specific binding” occurs between a pairedspecies where there is interaction between the two which produces abound complex having the characteristics of an antibody/antigen orenzyme/substrate interaction. In particular, the specific binding ischaracterized by the binding of one member of a pair to a particularspecies and to no other species within the family of compounds to whichthe corresponding member of the binding member belongs.

As used herein, the terms “Pan-DR epitopes,” “Pan-HLA-DR-bindingepitope,” “PADRE” and “PADRE peptides” mean a peptide of between about 4and about 20 residues that is capable of binding at least about 7 of the12 most common DR alleles (DR1, 2w2b, 2w2a, 3, 4w4, 4w14, 5, 7, 52a,52b, 52c, and 53) with high affinity. “High affinity” is defined hereinas binding with an IC₅₀% of less than 200 nm. For example, high affinitybinding includes binding with an IC₅₀% of less than 3100 nM. For bindingto Class II MHC, a binding affinity threshold of 1,000 nm is typical,and a binding affinity of less than 100 nm is generally considered highaffinity binding. Construction and use of PADRE peptides is described indetail in U.S. Pat. No. 5,736,142 which is incorporated herein byreference.

A “T helper peptide” as used herein refers to a peptide recognized bythe T cell receptor of T helper cells. For example, the PADRE peptidesdescribed herein are T helper peptides.

As used herein, the term “dendrimer” means a charged (e.g.,positively-charged, negatively-charged), highly branched polymericmacromolecule with roughly spherical shape. An example of apositively-charged, highly branched polymeric dendrimer is a PAMAMdendrimer. By the terms “PAMAM dendrimer” and “poly-amidoaminedendrimer” is meant a type of dendrimer in which tertiary amines arelocated at branching points and connections between structural layersare made by amide functional groups.

By the terms “PAMAM dendrimer” and “poly-amidoamine dendrimer” is meanta type of dendrimer in which tertiary amines are located at branchingpoints and connections between structural layers are made by amidefunctional groups. PAMAM dendrimers exhibit many positive charges ontheir surfaces.

By the term “derivatized dendrimer” is meant a dendrimer having one ormore functional groups conjugated to its surface.

A “PADRE-derivatized dendrimer” or “PADRE-dendrimer” is a nanoconstructin which one or more PADRE peptides are covalently attached to thefunctional groups on the surface of a charged (e.g., positively-charged)highly branched polymeric dendrimer (e.g., a PAMAM dendrimer).

By the term “conjugated” is meant when one molecule or agent isphysically or chemically coupled or adhered to another molecule oragent. Examples of conjugation include covalent linkage andelectrostatic complexation. The terms “complexed,” “complexed with,” and“conjugated” are used interchangeably herein.

As used herein, the phrase “sequence identity” means the percentage ofidentical subunits at corresponding positions in two sequences (e.g.,nucleic acid sequences, amino acid sequences) when the two sequences arealigned to maximize subunit matching, i.e., taking into account gaps andinsertions. Sequence identity can be measured using sequence analysissoftware (e.g., Sequence Analysis Software Package from Accelrys CGC,San Diego, Calif.).

The phrases “isolated” or biologically pure” refer to material which issubstantially or essentially free from components which normallyaccompany it as found in its native state.

As used herein, the term “nanoparticle” means a microscopic particlewhose size is measured in nanometers. For example, a nanoparticle is aPADRE-dendrimer conjugate or a particle combining severalPADRE-dendrimer conjugates and nucleic acid or amino acid material witha total diameter in the range of approximately 2-500 nm.

The term “antibody” is meant to include polyclonal antibodies,monoclonal antibodies (mAbs), chimeric antibodies, humanized antibodies,anti-idiotypic (anti-Id) antibodies to antibodies that can be labeled insoluble or bound form, as well as fragments, regions or derivativesthereof, provided by any known technique, such as, but not limited to,enzymatic cleavage, peptide synthesis or recombinant techniques.

As used herein the term “adjuvant” means any material which modulates toenhance the humoral and/or cellular immune response.

As used herein, the terms “displayed” or “surface exposed” areconsidered to be synonyms, and refer to antigens or other molecules thatare present (e.g., accessible to immune site recognition) at theexternal surface of a structure such as a nanoparticle (e.g.,PADRE-dendrimer).

By the term “multivalent” is meant that more than one copy or type ofantigen or molecule is displayed on a nanoparticle.

As used herein, “vaccine” includes all prophylactic and therapeuticvaccines. The vaccine compositions described herein are suitable foradministration to subjects in a biologically compatible form in vivo.The expression “biologically compatible form suitable for administrationin vivo” as used herein means a form of the substance to be administeredin which any toxic effects are outweighed by the therapeutic effects.The substances may be administered to any animal, e.g., humans. In someembodiments, a vaccine as described herein is administered to a mammal,e.g., a rodent or rabbit, for producing monoclonal antibodies against aparticular antigen.

By the phrase “immune response” is meant induction of antibody and/orimmune cell-mediated responses specific against an antigen or antigens.The induction of an immune response depends on many factors, includingthe immunogenic constitution of the challenged organism, the chemicalcomposition and configuration of the antigen, and the manner and periodof administration of the antigen. An immune response has many facets,some of which are exhibited by the cells of the immune system (e.g.,B-lymphocytes, T-lymphocytes, macrophages, and plasma cells). Immunesystem cells may participate in the immune response through interactionwith an antigen or other cells of the immune system, the release ofcytokines and reactivity to those cytokines. Immune responses aregenerally divided into two main categories—humoral and cell-mediated.The humoral component of the immune response includes production ofantibodies specific for an antigen. The cell-mediated component includesthe generation of delayed-type hypersensitivity and cytotoxic effectorcells against the antigen.

By the phrases “therapeutically effective amount” and “effective dosage”is meant an amount sufficient to produce a therapeutically (e.g.,clinically) desirable result; the exact nature of the result will varydepending on the nature of the disorder being treated. For example,where the disorder to be treated is cancer, the result can beelimination of cancerous cells including cancerous tumors. Thecompositions and vaccines described herein can be administered from oneor more times per day to one or more times per week. The skilled artisanwill appreciate that certain factors can influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof the compositions or vaccines of the invention can include a singletreatment or a series of treatments.

As used herein, the term “treatment” is defined as the application oradministration of a therapeutic agent described herein, or identified bya method described herein, to a patient, or application oradministration of the therapeutic agent to an isolated tissue or cellline from a patient, who has a disease, a symptom of disease or apredisposition toward a disease, with the purpose to cure, heal,alleviate, relieve, alter, remedy, ameliorate, improve or affect thedisease, the symptoms of disease, or the predisposition toward disease.

The terms “patient” “subject” and “individual” are used interchangeablyherein, and mean a mammalian subject to be treated, with human patientsbeing preferred. In some cases, the methods of the invention find use inexperimental animals, in veterinary applications, and in the developmentof animal models for disease, including, but not limited to, rodentsincluding mice, rats, and hamsters, as well as non-human primates.

Although vaccines, compositions, kits and methods similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable vaccines, compositions, kits and methods aredescribed below. All publications, patent applications, and patentsmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions, willcontrol. The particular embodiments discussed below are illustrativeonly and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pair of schematics showing the PADRE-dendrimer that may bemixed with plasmid or linked to a peptide or polypeptide antigen totarget APCs. FIG. 1 illustrates that the PADRE-dendrimers describedherein provide a platform in which any antigen of interest or nucleicacid encoding any antigen of interest can be incorporated. ThePADRE-dendrimers described herein are activators of innate immunity thatare designed to be grabbed by professional APCs.

FIG. 2 is a series of dot plot flow cytometry images of analysis ofhuman B cells showing in vitro delivery of PADRE-dendrimers complexedwith a short nucleic acid sequence tagged with a red fluorochrome. Thisnucleic acid is a red-labeled dsRNA oligomer designed for use in RNAianalysis to facilitate assessment and optimization of siRNAoligonucleotides delivery into mammalian cells. Cells were co-culturedwith the PADRE-dendrimers/multinucleotide complexes or controls for 4hours after which the media was removed and fresh media was added. Theimages show the delivery of dsRNA oligomer tagged with Alexa Fuor intopurified Human B cells. The lowest image in the fourth column of imagesshows the delivery of the oligo in approximately 92% of cells.

FIG. 3 a series of photographs showing in vivo DNA delivery ofPADRE-dendrimers. PADRE-dendrimer/GFP-plasmid complexes were injectedinto skin (5 μg total plasmid) and cornea (1 μg/cornea). Stereofluorescent microscope images were taken on live anesthetized mice. Theleft image shows the GFP expression in skin and the image on the rightshows GFP expression in the cornea.

FIG. 4 is a graph showing treatment of established tumors in mice. C57BLmice were immunized with plasmids encoding for either GFP (once) or OVA(twice) subcutaneously. The sera of three mice were collected and ELISA(left) or FLISA (right) were performed.

FIG. 5 is a pair of graphs showing PADRE-dendrimer therapy ofestablished tumors. Mice implanted with B16 melanoma cells (top) or TSA(bottom) were vaccinated on day two or three post-tumor implantationfollowed with booster immunizations after a week.

FIG. 6 is a series of flow cytometry histograms showing the expressionof GFP in human peripheral blood mononuclear cells (PBMC), lower panel,and in human B cells, upper panel, upon co-culturing GFP plasmid (5 μg)complexed with Dendrimer-PADRE. Dendrimer/GFP-plasmid complex was usedas a control, left histograms.

FIG. 7 is a series flow cytometry dot plots showing the in vitrodelivery of a protein, Albumin-FITC, into human B cells by PDD. The leftimages show PDD/Albumin-FITC delivery into purified human B cells. Humanpurified B cells were collected and were co-cultured withPDD/Albumin-FITC. The left histograms show the delivery of Albumin-FITCin human B cells the morning after the PDD/Albumin-FITC added to human Bcells. The Top histogram shows B cells alone, the histogram in theMiddle shows the Dendrime/Albumin-FITC complex plus B cells and thelower histogram depicts the results of PDD/Albumin-FITC complex added tohuman B cells. The right picture is the image of fluorescent microscopeof Albumin uptake by B cells one-hour post addition of PDD/Albumin-FITCcomplex.

FIG. 8 is a series of flow cytometry dot plots showing the in vivotargeting of DCs in the lymph node. The left image depicts a schematicof a timeline for injection and lymph node removal and analysis and theright image shows a pair of flow cytometry dot plots upon analysis ofdata obtained from cells of the lymph node adjacent to PDD/GFP-plasmidor Dendrimer/GFP-plasmid injection site versus a naive lymph node. Theseimages show the efficacy of in vivo PADRE-denhdrimer targeting of mouseDCs and B cells in an injection site neighboring the lymph node. Lymphcells were stained with CD11c (DC marker), MHC class II and CD20 (B cellmarker). The histograms in the right top show that Dendrimer/GFP-plasmidinjection resulted in the expression of GFP in approximately 6% of DCswhile the lower dot plot clearly shows that PDD/GFP-plasmid injectionresulted in the expression of GFP in >70% of DCs.

FIG. 9 is a pair of graphs showing that DRHA, a dendrimer decorated witha different T helper epitope, in vivo targeting DCs in the lymph nodeshows that DRHA facilitates GFP transfection into DCs. This experimentis similar to the one described in FIG. 8 with the difference thatBalb/c mice have been used in conjunction with dendrimer conjugated withIad-restricted HA peptide. The lymph node adjacent to theDRHA/GFP-plasmid or Dendrimer/GFP-plasmid injection site and a naivelymph node were removed on day 5 post-injection of DRHA/GFP-plasmid orDendrimer/GFP-plasmid. The charts show the results of the flow cytometryanalysis of data obtained from cells of the lymph node after stainingwith CD11c (DC marker) for DC. The top pane shows the number of DCpositive for GFP found draining lymph nodes of mice treated asindicated. The lower panel shows the mean fluorescence intensity of GFPwithin the DC. These results clearly indicate not only that DRHA augmentthe number of DC transfected in vivo but, also the number of plasmidmolecules that get into the cells.

FIG. 10 is a micrograph of human B cells transfected withPADRE-dendrimer complexed with a red(Alexa Fluor)-labeled dsRNA oligomeroligo

FIG. 11 is a pair of micrographs of PBMC of Baboon transfected withdendrimer complexed with a red(Alexa Fluor)-labeled dsRNA oligomer (leftpanel) and cells transfected with PADRE-dendrimer complexed with ared(Alexa Fluor)-labeled dsRNA oligomer (right panel). The flourescentmicroscope images were taken two hours post addition ofPDD/dsRNA-Alexa-Fluor or control complex to Baboon PBMC. The image showshigh efficacy of targeted delivery of multinucleutides to PBMC of monkeyvia PDD.

FIG. 12 is a pair of micrographs of Baboon PBMC transfected withdendrimer complexed with GFP-encoding plasmid (left panel) and cellstransfected with PADRE-dendrimer complexed with GFP-encoding plasmid(right panel). PBMC of Baboon transfected with dendrimer complexed withGFP-plasmid (left panel) and cells transfected with PADRE-dendrimercomplexed with GFP-plasmid (right panel). The flourescent microscopeimages were taken one day post addition of PDD/GFP-plasmid or controlcomplex to Baboon PBMC. The image shows high efficacy of targeteddelivery of the plasmid and the expression of the gene encoded by theplasmid via PDD.

FIG. 13 is a graph of results showing that a single DNA vaccination withPADRE-Dendrimer complexed with plasmid (DRP-ova plasmid) is superior toin vivo electroporation of plasmid (EP-ova plasmid).

FIG. 14 is pair of photographs of multi-well plates upon in-cell Westernassay using sera of immunized mice showing induction of high titresantibody responses in mice upon two immunizations with PDD/plasmid-PCARDantigen. Multi-well plates containeding cos-7 cells transfected withplasmid encoding antigen (left panel) and cos-7 cells transfected with acontrol plasmid (right panel).

FIG. 15 is a graph and a series of photographs of multi-well plates uponin-cell Western assay using sera of immunized mice showing induction ofhigh titres antibody responses in mice upon two immunizations withPDD/plasmid. Also shown are results from an in-cell Western FLISA afterone immunization with PADRE-dendrimers complexed with plasmids encodingeither GFP or ova or two immunizations with PADRE-dendrimers complexedwith plasmids encoding CCR5, vgPCR, CathL, or p2.

FIG. 16 is a pair of photographs of multi-well plates upon in-cellWestern assay using sera of immunized mice showing induction of antibodyresponses in mice upon one immunizations with PDD/plasmid-VgPCR that wasfurther mounted upon a second immunization with PDD/plasmid-VgPCR. Theseresults show that a single immunization with PDD/plasmid-VgPCR resultsin an antibody response which was enhanced upon a booster.

FIG. 17 is a graph showing UV-visible spectra of G5 dendrimer,conjugate, and peptide.

FIG. 18 is a graph showing eradication of B16/OVA tumors in aprophylactic setting by a vaccine as described herein(PADRE-dendrimer/OVA plasmid).

DETAILED DESCRIPTION

Described herein are nanoparticle-based vaccines, compositions, kits andmethods for effective delivery of one or more antigens in vivo forvaccination and antibody (e.g., monoclonal antibody) production, and forthe effective delivery of peptides, proteins, siRNA, RNA or DNA to PAPCsor MHC class II positive cells (e.g. tumor cells). In a typical vaccineor composition, a charged (e.g., positively-charged), highly branchedpolymeric dendrimer is conjugated to an MHC targeting and immunogenicpeptide such as a T helper peptide (e.g., an epitope such as the PADREpeptide or Influenza HA, etc.) and conjugated or bound to at least onemolecule for inducing an immune response to a particular antigen in asubject. The molecule may be a protein or peptide of bacterial, fungal,protozoan, or viral origin, or a fragment derived from these antigens, acarbohydrate, or a carbohydrate mimetic peptide. The molecule may alsoinclude self-antigens for the treatment of autoimmune diseases.Additionally, the antigenic molecule(s) may also include one or morenucleic acids including those in a mammalian plasmid encoding for atleast one antigen. For example, antigens may be one or more nucleicacids that result in expression of one or more immunogenic proteins andinduction of a robust and specific immune response to the expressedprotein(s) in a subject (e.g., mammal). As another example, antigens mayalso be immunogenic peptides or polypeptides that are processed andpresented. A charged (e.g., positively-charged), highly branchedpolymeric dendrimer can be conjugated to two or more different antigensand similarly, can be conjugated to two or more nucleic acids that eachencode a different antigen. A vaccine or other composition as describedherein can include a plurality of charged (e.g., positively-charged),highly branched polymeric dendrimers that are conjugated to one type ofantigen (e.g., five dendrimers conjugated to five copies of a particularantigen), or a plurality of charged (e.g., positively-charged), highlybranched polymeric dendrimers conjugated to a plurality of differentantigens (e.g., five dendrimers conjugated five different antigens). Thedendrimer makes a complex (conjugation) with antigens (nucleic acids orproteins) based on the opposite charge of the dendrimer (positive) andthat of antigen (negative) or the conjugation may be a covalent chemicallinkage.

In one embodiment, a nanoparticle-based method to deliver antigens invivo as described herein includes injection of a vaccine composed of aDNA plasmid encoding an antigen bound to, or an antigenic peptide orpolypeptide conjugated to a charged (e.g., positively-charged), highlybranched polymeric dendrimer (e.g., PADRE-derivatized dendrimer (PDD))that is also conjugated to an MHC targeting and immunogenic peptide suchas a T helper peptide (e.g., an epitope such as the PADRE peptide orInfluenza HA, etc.). Negatively-charged plasmids bind naturally to thepositively-charged PADRE-dendrimers, while peptide or polypeptideantigens can be chemically linked to the PADRE-dendrimers if they arenot negatively-charged. In other embodiments, a dendrimer isnegatively-charged for binding to positively-charged proteins andpeptides. Surface-exposed antigen(s) or nucleic acid(s) encoding anantigen(s) may be conjugated to the dendrimers by any suitable meansknown in the art. Conjugation methods include chemical complexation,which may be either ionic or non-ionic in nature, electrostatic binding,or covalent binding. A dendrimer conjugated to a T helper epitope asdescribed herein can be multivalent; it can present more than one copyor type of antigen or nucleic acid on its surface. Presentation ofmultivalent or aggregated antigens (or nucleic acids encoding antigens)may improve the immune response of a subject. The one or more copies ortypes of antigens or nucleic acids can be attached to the dendrimer viatwo or more separate linkers or spacers, or via a common linker orspacer. The compositions, kits and vaccines described herein have bothprophylactic and treatment applications, i.e., can be used as aprophylactic to prevent onset of a disease or condition in a subject, aswell as to treat a subject having a disease or condition. A vaccine asdescribed herein can be used to mount an immune response against anyinfectious pathogen or cancer.

The therapeutic agents described herein can be used to targetmononuclear cells, in particular B cells, and can be used to treatconcurrent B-cell chronic lymphocytic leukemia, and Multiple myeloma. Acombination of nanoparticle as described herein (e.g., PADRE-derivatizeddendrimer) and therapeutic agent (e.g., drug) may be used in severalforms, e.g., a mixture of the nanoparticle with the therapeutic agent,electrostaticlly bound to form a complex, chemical conjugation of thetherapeutic agent to the nanoparticle, etc. Examples of therapeuticagents includes but are not limited to toxins, iRNA, siRNA, microRNA,plasmid (e.g., encoding tumor suppressor genes, suicide genes (e.g., TK)or any genes the block or alter tumor proliferation and/or survival),Taxol® (paclitaxel) (Bristol-Myers Squibb), antibodies, melphalan,prednisone, thalidomide (MPT), Velcade® (bortezomib) (MilleniumPharmaceuticals), lenalidomide, and dexamethasone or any combination ofsuch agents.

Similarly, the compositions and methods described herein may be used forthe therapy of autoimmune disorders, where the therapeutic agent reaches(be delivered to) immune cells including monocytes, DCs, T cells or Bcells. The compositions described herein may be used with adjuvants suchas (but not limited to) Poly I:C which is negatively charged and makes acomplex with the nanoparticle platform as described herein.

The below described preferred embodiments illustrate adaptations ofthese compositions, vaccines, kits and methods. Nonetheless, from thedescription of these embodiments, other aspects of the invention can bemade and/or practiced based on the description provided below.

Biological Methods

Methods involving conventional molecular biology techniques aredescribed herein. Such techniques are generally known in the art and aredescribed in detail in methodology treatises such as Molecular Cloning:A Laboratory Manual, 3rd ed., vol. 1-3, ed. Sambrook et al., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and CurrentProtocols in Molecular Biology, ed. Ausubel et al., Greene Publishingand Wiley-Interscience, New York, 1992 (with periodic updates).Immunology techniques are generally known in the art and are describedin detail in methodology treatises such as Advances in Immunology,volume 93, ed. Frederick W. Alt, Academic Press, Burlington, Mass.,2007; Making and Using Antibodies: A Practical Handbook, eds. Gary C.Howard and Matthew R. Kaser, CRC Press, Boca Raton, Fla., 2006; MedicalImmunology, 6^(th) ed., edited by Gabriel Virella, Informa HealthcarePress, London, England, 2007; and Harlow and Lane ANTIBODIES: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988. Conventional methods of gene transfer and genetherapy may also be adapted for use in the present invention. See, e.g.,Gene Therapy: Principles and Applications, ed. T. Blackenstein, SpringerVerlag, 1999; and Gene Therapy Protocols (Methods in MolecularMedicine), ed. P. D. Robbins, Humana Press, 1997. Methods of vaccineproduction and administering vaccines are also generally known in theart and are described in detail, for example, in Vaccine Protocols(Methods in Molecular Medicine) by Andrew Robinson, Martin P. Cranage,and Michael J. Hudson, 2nd ed., Humana Press, Totowa, N.J., 2003;Vaccine Adjuvants and Delivery Systems, by Manmohan Singh, 1st ed.,Wiley-Interscience, Hoboken, N.J., 2007; Arvin A. M. and Greenberg H.B., Virology 344:240-249, 2006; and R. Morenweiser, Gene Therapy suppl.1:S103-S110, 2005. Construction and use of vaccines as well as PAMAMdendrimers is also described, for example, in Arashkia et al., VirusGenes 40 (1): 44-52, 2010; Velders et al., J Immunol. 166:5366-5373,2001; and S. Chauhan, N. K. Jain, P. V. Diwan. (2009) Pre-clinical andbehavioural toxicity profile of PAMAM dendrimers in mice. Proceedings ofthe Royal Society A: Mathematical, Physical and Engineering Sciences(Online publication date: Dec. 3, 2009).

Synthesis of Dendrimers Conjugated to Nucleic Acids, Peptides orPolypeptides

Dendrimers act as scaffolds to condense DNA, and a fullypositively-charged dendrimer is preferable for developing strongelectrostatic interactions with a negatively-charged DNA or RNA. Aresulting dendrimer/T helper epitope/DNA complex, for example, has a netcharge depending on the adjustable N/P ratio (amine to phosphate orcharge ratio). Described herein are dendrimers having conjugated theretoT helper peptides (e.g., an epitope such as the PADRE peptide orInfluenza HA) and an antigen, a nucleic acid encoding an antigen, or ansiRNA, wherein the at least one T helper peptide and the antigen,nucleic acid or siRNA are conjugated to the exterior surface of thedendrimer such that the at least one T helper peptide specifically bindsto PAPCs. In one embodiment, dendrimers are conjugated to at least onePADRE peptide (e.g., 2, 3, 4, 5, etc.) and a peptide or polypeptideantigen. In this embodiment, a dendrimer is typically conjugated to orbound to (e.g., via an electrostatic binding) to a plurality of thepeptide or polypeptide antigen. Conjugating or binding several antigens(e.g., a plurality of the same antigen) may be particularly useful whenthe antigen is a small antigen (especially small peptides orcarbohydrates), as small antigens generally fail to elicit an effectiveimmune response due to hapten-related size issues. Including multiplecopies of an antigen into the dendrimer/T helper peptide/nucleic acidconjugates described herein can thus enhance the immunogenicity of theantigen. In another embodiment, dendrimers are conjugated to PADREpeptides and bound to a nucleic acid encoding an antigen. In yet anotherembodiment, dendrimers are conjugated to PADRE peptides and bound to ansiRNA directed against a gene of interest. In these embodiments, thedendrimers can be prepared and conjugated to a T helper peptide (e.g.,an epitope such as the PADRE peptide or Influenza HA) and bound tonucleic acid (e.g., DNA, siRNA) or peptide or polypeptide using anysuitable method. Methods of producing and using dendrimers are wellknown in the art and are described, for example, in Zhang J-T et. al.Macromol. Biosci. 2004, 4, 575-578, and U.S. Pat. Nos. 4,216,171 and5,795,582, both incorporated herein by reference. See also: D. A.Tomalia, A. M. Naylor, and W. A. Goddard III, “Starburst Dendrimers:Molecular-Level Control of Size, Shape, Surface Chemistry, Topology, andFlexibility from Atoms to Macroscopic Matter”, Angew. Chem. Int. Ed.Engl. 29 (1990), 138-175. In the experiments described herein, PAMAMdendrimers were used. However, any suitable positively charged, highlybranched polymeric dendrimer can be used. Examples of additionalpositively charged, highly branched polymeric dendrimers includepoly(propylene imine) (PPI) dendrimers or, more generally, any otherdendrimers with primary amine groups on their surfaces.

The PADRE-dendrimers (PADRE-derivatized dendrimers) described herein canbe prepared by any suitable method. Methods of making and using PADREare known in the art. See, for example, U.S. Pat. No. 5,736,142. Toproduce the PADRE peptides described in U.S. Pat. No. 5,736,142, astrategy initially described by Jardetzky et al. (EMBO J. 9:1797-1083,1990) was used, in which anchor residues that contain side chainscritical for the binding to MHC are inserted into a poly-alanine peptideof 13 residues. PADRE peptides can be prepared according to the methodsdescribed in U.S. Pat. No. 5,736,142, for example, or they can bepurchased (e.g., from Anaspec, Inc., Fremont, Calif.). Because of theirrelatively short size, the PADRE peptides can be synthesized in solutionor on a solid support in accordance with conventional techniques.Various automatic synthesizers are commercially available and can beused in accordance with known protocols. Alternatively, recombinant DNAtechnology may be employed wherein a nucleotide sequence which encodes aT helper epitope is inserted into an expression vector, transformed ortransfected into an appropriate host cell and cultivated underconditions suitable for expression. These procedures are generally knownin the art, as described generally in Sambrook et al., (supra), which isincorporated herein by reference. PADRE peptides as described herein mayinclude modifications to the N- and C-terminal residues. As will be wellunderstood by the artisan, the N- and C-termini may be modified to alterphysical or chemical properties of the peptide, such as, for example, toaffect binding, stability, bioavailability, ease of linking, and thelike. The PADRE peptides described herein may be modified in any numberof ways to provide desired attributes, e.g., improved pharmacologicalcharacteristics, while retaining substantially all of the biologicalactivity of the unmodified peptide.

In the experiments described herein, the PADRE-dendrimer conjugate wasmade by simple amide coupling between the —COOH terminus of the PADREpeptide and one of the dendrimer amine groups. The PADRE peptide(Ac-D-Ala-Lys-Cha-Val-Ala-Ala-Trp-Thr-Leu-Lys-Ala-Ala-Ala-D-Ala-Ahx-Cys-OH)(SEQ ID NO:1, Ac=acetylated; D-Ala=D-alanine; Cha=cyclohexylalanine;Ahx=aminohexanoic acid) was purchased from Anaspec, Inc., (Fremont,Calif.) in its acetylated form in order to protect the amine terminusand prevent its reaction. The purchased peptide had a minimum purity of95%. The amide coupling reaction was carried out under standardconditions (see FIG. 1, bottom schematic) in DMF solution. In order tocontrol the number of PADRE epitopes attached to the surface of eachdendrimer, a 2:1 peptide/dendrimer challenge ratio was used in thereaction, seeking attachment of just a few peptides per dendrimer inorder to keep most of the amine groups free to develop large positivecharges on the dendrimer. In a typical embodiment, a plurality ofPADRE-dendrimer conjugates as described herein will be a distribution ofdendrimers containing 0, 1, 2, 3, etc., PADREs (or other peptide)attached thereto. Relative populations are expected to follow thePoisson distribution. The PADRE, aKXVAAWTLKAAa (SEQ ID NO:2) binds withhigh or intermediate affinity (IC₅₀<1,000 nM) to 15 out of 16 of themost prevalent HLA-DR molecules ((Kawashima et al., Human Immunology59:1-14 (1998); Alexander et al., Immunity 1:751-761 (1994)). However,other peptides which also can bind MHC class II and activate CD4 Thelper cells in most humans may also be used to tag the dendrimer.

Examples of peptides include but are not limited to: tetanus toxoid (TT)peptide 830-843; the “universal” epitope described in Panina-Bordignonet al., (Eur. J. Immunology 19:2237-2242 (1989)); and the followingpeptides that react with MHC class II of most human HLA, and many ofmice: aKFVAAWTLKAAa (SEQ ID NO:3), aKYVAAWTLKAAa (SEQ ID NO:4),aKFVAAYTLKAAa (SEQ ID NO:5), aKXVAAYTLKAAa (SEQ ID NO:6), aKYVAAYTLKAAa(SEQ ID NO:7), aKFVAAHTLKAAa (SEQ ID NO:8), aKXVAAHTLKAAa (SEQ ID NO:9),aKYVAAHTLKAAa (SEQ ID NO:10), aKFVAANTLKAAa (SEQ ID NO:11),aKXVAANTLKAAa (SEQ ID NO:12), aKYVAANTLKAAa (SEQ ID NO:13),AKXVAAWTLKAAA (SEQ ID NO:2), AKFVAAWTLKAAA (SEQ ID NO:14), AKYVAAWTLKAAA(SEQ ID NO:15), AKFVAAYTLKAAA (SEQ ID NO:16), AKXVAAYTLKAAA(SEQ IDNO:17), AKYVAAYTLKAAA (SEQ ID NO:18), AKFVAAHTLKAAA (SEQ ID NO:19),AKXVAAHTLKAAA (SEQ ID NO:20), AKYVAAHTLKAAA (SEQ ID NO:21),AKFVAANTLKAAA (SEQ ID NO:22), AKXVAANTLKAAA (SEQ ID NO:23), andAKYVAANTLKAAA (SEQ ID NO:24) (a=D-alanine, X=cyclohexylalanine). Anotherexample of an epitope that may be used is the HA peptide sequenceSFERFEIFPKE (SEQ ID NO:25) (from the provirus PR8 virus HA) that bindsto mouse Balb/c MHC classII IaD.

The product was purified by dialysis against pure water for at least 24h and then dried under vacuum. The collected product, a clear oil, wascharacterized by ¹H NMR, UV-Vis and MALDI-TOF mass spectroscopy. The NMRspectra of the PADRE-dendrimer conjugate shows large peaks correspondingto the dendrimer protons and a small set of peaks for the peptideprotons. The MALDI-TOF mass spectrum of the PADRE-dendrimer conjugateshows a peak at a m/z ratio ca. 3,000 units higher than the peakobserved for the dendrimer on its own. The excess mass corresponds toapproximately 2 peptide epitopes. The UV-Vis spectrum of the conjugateshows a clear absorption in the wavelength range where tryptophanabsorbs.

Complexation of plasmid DNA with the PADRE-dendrimer conjugate was doneby mixing the two components in aqueous solution buffered atphysiological pH with PBS. Typical N/P (amine to phosphate) ratios are10:1. Gel electrophoresis is used to show complete complexation of theDNA. At physiological pH values, the amino groups (—NH₂) are protonated,affording a high positive charge to the dendrimers and making themparticularly well-suited for the delivery of negatively-charged DNA orRNA into cells. In aqueous solution, the positively-charged dendrimersand the negatively-charged nucleic acids give rise to condensates ornanoparticles which can penetrate and traverse biological membranes withrelative ease.

Dendrimers that are conjugated to T helper epitopes other than PADRE aretypically prepared by a method similar to that described above forPADRE-derivatized dendrimers. For example, the acid terminus of thepeptide can be covalently attached to one of the amine groups on thedendrimer surface by a number of well-known synthetic methods, such asamidation using carbodiimides as activating reagents As another example,attachment of these peptides to amino-terminated dendrimers is performedusing two synthetic routes. The amino terminus of the peptide epitope isprotected by acetylation. The first route uses the carboxylic acid ofthe terminal cysteine residue to achieve attachment via standardamidation chemistry. The second route takes advantage of the cysteine'sthiol (if present on the peptide, otherwise may be added) to react itwith the alkene groups added to the dendrimer surface by previoustreatment with maleimide. Both routes allow the functionalization ofdendrimers with epitopes. Up to several peptide epitopes (e.g., 2, 3, 4,5, 6, etc.) per dendrimer will enhance the targeting property of the DNAdelivery agents, improving their properties for vaccination purposes.However, it is important to leave a large number of unreacted aminegroups so that the dendrimer will acquire a large positive charge viaprotonation at physiological pH values. Dendrimers as described hereincan be conjugated to any T helper epitope. An example of an additional Thelper epitope is Influenza HA.

Generally, generation-5 (G5) dendrimers are used in the compositions,kits and methods described herein. However, other generation dendrimers(see Table 1) can be used.

TABLE 1 PAMAM Dendrimers Generation Molecular Weight Diameter (nm)Surface Groups 0 517 1.5 4 1 1,430 2.2 8 2 3,256 2.9 16 3 6,909 3.6 32 414,215 4.5 64 5 28,826 5.4 128 6 58,0548 6.7 256

Charged Polymeric Carrier Vaccines and Compositions

A vaccine as described herein includes at least one charged (e.g.,positively-charged) polymeric carrier such as a dendrimer havingconjugated or bound thereto an MHC targeting and immunogenic peptidesuch as a T helper peptide (e.g., an epitope such as the PADRE peptideor Influenza HA) and at least one peptide or polypeptide antigen or atleast one nucleic acid encoding the at least one antigen such that theat least one MHC targeting and immunogenic peptide and the at least onenucleic acid or at least one peptide or polypeptide antigen areconjugated to the exterior surface of the charged (e.g.,positively-charged) polymeric carrier (e.g., dendrimer) and the MHCtargeting and immunogenic peptide (e.g., T helper epitope) specificallybinds to PAPCs. The combination of the at least one T helper peptide, atleast one dendrimer and at least one peptide or polypeptide antigen orat least one nucleic acid encoding the at least one antigen are able toinduce an immune response against the at least one antigen includinginduction of MHC class II mediated activation of helper T cells. Avaccine may further include a water-in-oil emulsion. Administering thevaccine to the mammal results in production of monoclonal antibodiesagainst the antigen. Antigen or antigens as described herein that aredisplayed on or within the dendrimers induce an immune response againstonset of disease caused by a variety of pathogenic conditions. In oneembodiment, the antigen may be derived from, but are not limited to,pathogenic bacterial, fungal, or viral organisms, Streptococcus species,Candida species, Brucella species, Salmonella species, Shigella species,Pseudomonas species, Bordetella species, Clostridium species, Norwalkvirus, Bacillus anthracis, Mycobacterium tuberculosis, humanimmunodeficiency virus (HIV), Chlamydia species, human Papillomaviruses,Influenza virus, Paramyxovirus species, Herpes virus, Cytomegalovirus,Varicella-Zoster virus, Epstein-Barr virus, Hepatitis viruses,Plasmodium species, Trichomonas species, sexually transmitted diseaseagents, viral encephalitis agents, protozoan disease agents, fungaldisease agents, bacterial disease agents, cancer cells, or mixturesthereof.

The at least one dendrimer can be further conjugated toPolyinosinic-polycytidylic acid (Poly(I:C)), and the vaccine orcomposition can further include a pharmaceutically acceptable carrier.In one embodiment, the at least one T helper epitope is a Pan-DRepitope, e.g., two Pan-DR epitopes each having the amino acid sequenceof SEQ ID NO:1. In another embodiment, the T helper epitope is influenzaHA. The T helper epitope, however, can be any epitope that activates orcontributes to activation of CD4+T helper cells. T helper epitopeactivation of CD4+T helper cells is required for the expansion andstimulation of CD8 T cells as well as for antibody production by Bcells, both of which are essential for induction of protective immuneresponses against infectious agents or cancer. In an embodiment in whichthe dendrimer is conjugated to a nucleic acid encoding an antigen, thenucleic acid is generally an expression vector. The expression vectortypically includes a eukaryotic promoter operably linked to a geneencoding the antigen, a cloning site, a polyadenylation sequence, aselectable marker and a bacterial origin of replication. Generally, theantigen is typically a cancer antigen or an antigen from an infectiouspathogen. The at least one dendrimer is generally a G5 dendrimer.Similarly, in embodiments in which the dendrimer is conjugated to apeptide or polypeptide antigen, the antigen is generally a cancerantigen or an antigen from an infectious pathogen, and the at least onedendrimer is a G5 dendrimer. In some embodiments, an adjuvant may beincorporated in the vaccine or composition.

Dendrimers are effective vehicles to escort DNA (and other nucleic acidsincluding DNA, RNA, siRNA, microRNA, RNAi, etc.) into cells. However, asa vaccine delivery platform, dendrimers have traditionally been afailure for several reasons. First, dendrimers lack adjuvant activity;they lack effective stimulation of innate immunity, and they do notgenerate a “danger signal”. Also, dendrimers provide poor targeting ofAPCs and in general, they provide poor stimulation of adaptive immunity.A robust adjuvanted vaccine delivery system that specifically targetsPAPCs, that induces a “danger signal,” that recruits professionalmononuclear cells to injection sites, and that is safe is highlydesired, particularly when dealing with poor antigens with lowimmunogenicity or “self” antigens or those with high homology with“self” antigens against which the immune system has developed tolerance.Poor antigens or those with low immunogenicity result in no or lowlevels of specific immune responses, antibody responses or cell-mediatedimmune responses. The vaccine platform described herein is abiodegradable nanoparticle complexed with (conjugated to) DNA or apeptide or polypeptide antigen. The platform targets PAPCs via its MHCclass II ligand, binds and penetrates the cell membrane by its highlypositively-charged outer membrane, is safe and easy to scale up forhigh-volume production, and acts as a strong adjuvant due to the natureof modifications on the molecule. In a typical embodiment, the at leastone dendrimer is a G5 PAMAM dendrimer that is a highly branchedpolymeric macromolecule and an ideal excipient for its enhancedsolubility. G5 is, in particular, ideal for the delivery of DNA intocells. A typical vaccine as described herein includes a water-in-oilemulsion that induces a transient danger signal resulting in therecruitment of mononuclear cells to the injection site. These cells,upon picking up the DNA, will travel to regional lymph nodes and presentantigen. Inclusion of a universal T helper agonist, e.g., PADRE, whichbinds to the flank of the MHC class II molecules, results in anopsinizing dendrimer complex for PAPCs as well as helper T cells. Thisalteration changes an inert and weak dendimer to a robust immuneenhancer for the expressed antigen of interest. Also including ahydrophobic career, i.e., an oil emulsion (e.g., Montanide ISA 720),that has both adjuvant activity as well as a depot effect, results in aslow release of antigen. Inclusion of poly(I:C) further enhancesinduction of an immune response against an antigen of interest. Sincepoly(I:C) has a negative net charge, it conveniently binds to dendrimer,and it is an adjuvant that enhances the robustness of an immuneresponse. These features act as strong “danger signals” and recruitfurther mononuclear cells (including APCs) to the injection site.Collectively, these features stimulate innate immunity and result inenhanced expression of a proinflammatory cytokine milieu needed forinducing effective immune responses.

In one embodiment of a vaccine, the T helper peptide is a PADRE epitopeand the dendrimer is PADRE-derivatized. PADRE is an artificiallydesigned peptide that binds to the majority of MHC Class II, andconjugating PADRE peptides to dendrimers (e.g., a PADRE-derivatizeddendrimer) makes the resultant complex or conjugate a ligand for PAPCsthat express high levels of MHC class II. This complex thus becomes auniversal targeted vaccine delivery system with high affinity for cellsexpressing MHC class II or PAPCs. PADRE also activates T helper cellsand results in a milieu of proinflammatory cytokines, and recruits otherimmune cells to the injection site. Combined with a dendrimer, PADREfurther enhances the uptake of antigen by inducing a “danger signal.” APADRE-derivatized dendrimer provides several advantages over currentlyknown vaccines. First, a G5 dendrimer is a highly charged biodegradablemolecule that will bind and enter a cell membrane very efficientlyresulting in robust expression of the protein. Second, PADRE is auniversal T-helper epitope that binds to many murine and human MHC classII molecules. It is a synthetic, non-natural T helper epitope[AKchxAVAAWTLKAAA (SEQ ID NO:26) (chxA=cyclohexylalanine)]. When fusedto the surface of the dendrimer, PADRE will bind and activate primarilycells that have MHC class II including all PAPCs. Several PADRE epitopes(e.g., 2, 3, 4, 5, etc.) can be attached to each dendrimer. Theattachment is done with suitable spacers to preserve the bindingproperties of the peptide that give rise to its immunogenic properties.A linker or spacer molecule may be used in conjugating antigen or othermolecules to the dendrimer conjugates described herein. Spacers may beany combination of amino acids including AAA, KK, GS, GSGGGGS (SEQ IDNO:27), RS, or AAY. As used herein, the terms “linker” or “spacer” meanthe chemical groups that are interposed between the dendrimer and thesurface exposed molecule(s) such as the MHC class II ligand, CD4+Thelper epitope, polypeptide, or therapeutic agent that is conjugated orbound to the dendrimer (e.g., PADRE-dendrimer) and the surface exposedmolecule(s). Preferably, linkers are conjugated to the surface moleculeat one end and at their other end to the nanoparticle (e.g.,PADRE-dendrimer). Linking may be performed with either homo- orheterobifunctional agents, i.e., SPDP, DSS, SIAB. Methods for linkingare disclosed in PCT/DK00/00531 (WO 01/22995) to deJongh, et al., whichis hereby incorporated by reference in its entirety.

Third, in embodiments in which the vaccine also includes a water-in-oilemulsion, the water-in-oil emulsion induces a transient “danger signal”resulting in the recruitment of mononuclear cells to the injection site.These cells, upon picking up the DNA, will travel to regional lymphnodes and present antigen. In addition, to further enhance induction ofan immune response, a synthetic double-stranded RNA (dsRNA), poly(I:C),can be bound to the dendrimer (e.g., a PADRE-dendrimer). Poly(I:C) is aToll-like receptor 3 (TLR-3) agonist that has a negative net charge andthus conveniently binds to dendrimer. Poly(I:C) is an adjuvant and dueto its negative net charge, will bind to the dendrimer and enhancerobustness of the immune response. Binding Poly(I:C) to dendrimers is ofparticular use for the production of monoclonal antibodies (discussedbelow) because it reduces the frequency of and intervals betweeninjections.

Nucleic acid molecules encoding an antigen as described herein may be inthe form of RNA (e.g., mRNA, microRNA, siRNA, shRNA or syntheticchemically modified RNA) or in the form of DNA (e.g., cDNA, genomic DNA,and synthetic DNA). The DNA may be double-stranded or single-stranded,and if single-stranded, may be the coding (sense) strand or non-coding(anti-sense) strand. In one embodiment, a nucleic acid can be an RNAmolecule isolated or amplified from immortalized or primary tumor celllines.

As described above, in one embodiment, a vaccine for inducing an immuneresponse includes at least one dendrimer having conjugated thereto atleast one T helper epitope and a nucleic acid encoding an antigen,wherein the resultant complex induces an immune response against theantigen. These compositions and methods are far safer, simpler and rapidcompared to other genetic immunization methods that require the use ofviral vectors or in vivo electroporation, for example. The use of DNAfor the induction of humoral or cellular immune responses has severaladvantages. First, use of DNA provides a full spectrum of naïve(naturally) processed epitopes. Also, dendrimers conjugated to a Thelper epitope and a nucleic acid encoding an antigen provide auniversal vaccine delivery targeted to APCs of >95% of all human MHCs(AKA, HLA) and eliminate the need for the purification of proteins thatare challenging to purify. Such proteins can be part of a multi-proteincomplex, can be membrane proteins, and can be incorrectly folded andinsoluble. The dendrimer conjugates described herein do not requireglycoslyation or posttranslational modifications of proteins, they taginterference with protein structure or folding, and offer dramatic costand time savings. The fact that PADRE-dendrimer targets and deliversnucleic acids to PBMC from mice, Baboons and humans makes this platforman ideal candidate for rapid translational research from mice tonon-human primates, and humans.

Also as described above, in another embodiment, a vaccine for inducingan immune response includes a water-in-oil emulsion and at least onedendrimer having conjugated thereto at least one T helper peptide (e.g.,an epitope such as the PADRE peptide or Influenza HA) and a peptide orpolypeptide antigen, wherein the at least one T helper epitope and thepeptide or polypeptide antigen are conjugated to the exterior surface ofthe dendrimer and are able to induce an immune response against thepeptide or polypeptide antigen. Peptides or polypeptides that have weakimmunogenicity induce robust immune responses when conjugated to(complexed-with) T helper epitope/dendrimer complexes as describedherein. Polypeptides and peptides with a negative net charge may complexwith, for example, PADRE-dendrimer with no need for covalentconjugation. A water-in-oil emulsion of, for example, a PADRE-dendrimerresults in further adjuvant activity and a depot effect.

As mentioned above, the compositions and vaccines described herein haveboth prophylactic and treatment applications; they can be used as aprophylactic to prevent onset of a disease or condition in a subject, aswell as to treat a subject having a disease or condition. A vaccine asdescribed herein can be used to mount an immune response against anyinfectious pathogen or cancer. Examples of infectious pathogens includeviruses such as, but not limited to, influenza, HIV, dengue virus,rotavirus, HPV, HBV, HCV, CMV, HSV, HZV, and EBV, pathogenic agentsincluding the causative agents of Malaria, Plasmodium(p) falciparum, P.malariae, P. ovale, P. vivax and P. knowlesi; the casatve agent ofLeishmania (L), L. major, L. tropica, L. aethiopica, L. mexicana, L.donovani, L. infantum syn. L. chagas; pathogenic bacteria includingBacillus anthracis, Bordetella pertussis, Streptococcus pneumonia, andmeningococcus. In the experiments described herein, PADRE-dendrimerseradicated established melanoma tumors in mice. However, the dendrimersconjugated to a T helper epitope and an antigen or a nucleic acidencoding an antigen as described herein can be used to mount a specificimmune response against any cancer. Examples of additional cancersinclude HPV-induced cervical cancers (e.g., E7/E7 tumor associatedantigens (TAA) or plasmids encoding for these antigens can be complexedwith the T helper epitope/dendrimers (e.g. PADRE-dendrimer) describedherein), human melanoma (e.g., TRP-1, TRP-2, gp-100, MAGE-1, MAGE-3and/or p53 may be used as TAA and complexed with the T helperepitope/dendrimers (e.g. PADRE-dendrimer) described herein), andprostate cancer (e.g., TSA may be used as TAA and complexed with the Thelper epitope/dendrimers (e.g. PADRE-dendrimer) described herein).Similarly for lung tumors, breast tumors, and leukemia, any suitable TAAcan be used, and many have been described. Many such TAA are commonbetween various cancers (e.g., CEA, MUC-1, Her2, CD20). A cocktail ofTAA or plasmids encoding for such antigens may be used to make auniversal, multiple-use cancer vaccine as described herein. In oneexample of a vaccine as described herein, a CD4 epitope/dendrimer (e.g.,PADRE-dendrimer), may be complexed with more than one antigen or withmore than one plasmid encoding the antigen. Alternatively, multiplevaccines each complexed with one antigen or with one plasmid encodingfor one antigen may be mixed and used as one vaccine for variouspathogens or various cancers.

Methods of Delivering an Antigen to a Mammal and Inducing an ImmuneResponse

Described herein are methods of delivering an antigen to a mammal (e.g.,human) and inducing production of monoclonal antibodies against theantigen for inducing a immune response in the mammal. A typical methodincludes the steps of: administering to the mammal a compositionincluding at least one charged (e.g., positively-charged) polymericcarrier (e.g., a dendrimer) having conjugated thereto an MHC targetingand immunogenic peptide (e.g., a T helper peptide such as the PADREpeptide or Influenza HA, etc.) and at least one peptide or polypeptideantigen or at least one nucleic acid encoding the at least one antigenwherein the at least one T helper peptide and the at least one nucleicacid or at least one peptide or polypeptide antigen are conjugated tothe exterior surface of the at least one charged (e.g.,positively-charged) polymeric carrier (e.g., dendrimer) such that the atleast one MHC targeting and immunogenic peptide (e.g., T helper epitope)specifically binds to PAPCs and the combination of the at least one MHCtargeting and immunogenic peptide (e.g., T helper epitope), at least onecharged (e.g., positively-charged) polymeric carrier (e.g., dendrimer),and the at least one nucleic acid or least one peptide or polypeptideantigen are able to induce an immune response against the antigen. Inthe method, the composition is administered in an amount effective toinduce MHC class II mediated activation of helper T cells, resulting inproduction of monoclonal antibodies and an immune response against theantigen in the mammal. A composition can further include a water-in-oilemulsion. The at least one dendrimer is typically further conjugated topoly(I:C), and the composition typically further includes apharmaceutically acceptable carrier. The at least one T helper epitopecan be a Pan-DR epitope, e.g., two Pan-DR epitopes each having the aminoacid sequence of SEQ ID NO:1. Alternatively, the at least one T helperepitope can be other than a Pan-DR epitope (PADRE epitope), e.g.,influenza HA. Generally, the at least one dendrimer is a G5 dendrimer.

In one embodiment, the mammal has cancer, the antigen is a cancerantigen, and the composition is a vaccine for the cancer. In anotherembodiment, the mammal has an infectious disease, the antigen is anantigen from an infectious pathogen, and the composition is a vaccinefor the infectious pathogen. In such embodiments, administration of thecomposition generally results in no local adverse reactions in themammal. Such methods are generally performed by formulating thecomposition (e.g., vaccine) outside of the mammal and administering thecomposition to the mammal in an amount sufficient to stimulate an immuneresponse against the antigen, e.g., a cancer antigen or antigen from aninfectious pathogen, in the mammal. The compositions, vaccines andmethods described herein can be utilized with any suitable subject,e.g., an animal such as a mammal (e.g., human beings, rodents, dogs,cats, goats, sheep, cows, horses, etc.). A human patient suffering fromor at risk for developing a cancer or infectious disease is a typicalsubject.

Compositions and Methods for Delivering siRNA to PAPCs

Also described herein are compositions and methods for delivering ansiRNA into a PAPC. In a typical embodiment, a composition for deliveringan siRNA into a PAPC includes at least one at least one charged (e.g.,positively-charged) polymeric carrier (e.g., a dendrimer) havingconjugated thereto at least one MHC targeting and immunogenic peptide(e.g., a T helper peptide) and an siRNA. The at least one MHC targetingand immunogenic peptide (e.g., T helper epitope) and the siRNA areconjugated to the exterior surface of the at least one charged (e.g.,positively-charged) polymeric carrier (e.g., dendrimer) such that the atleast one T helper epitope specifically binds to PAPCs. The siRNA can bedirected against (specific for) any gene of interest (e.g., Foxp3,CD-28, CTLA-4). The composition can further include a water-in-oilemulsion.

A typical method of delivering siRNA into PAPCs includes the steps of:providing a composition including at least one dendrimer havingconjugated thereto at least one T helper peptide and at least one siRNA,wherein the at least one T helper peptide and the at least one siRNA areconjugated to the exterior surface of the dendrimer such that the atleast one T helper peptide specifically binds to PAPCs; andadministering the composition to a mammalian subject under conditions inwhich the at least one dendrimer having conjugated thereto at least oneT helper peptide and at least one siRNA binds to a PAPC and the siRNAenters the PAPC and is expressed within the PAPC. The composition canfurther include a water-in-oil emulsion. In the method, the siRNA can bedirected against (specific for) any gene of interest (e.g., FOXp3) tosilence the expression of the gene of interest. In one example, siRNAdirected against FoxP3 is used to silence expression of FoxP3, amolecule that results in induction of regulatory T cells, cells thatsuppress immune responses and act as a negative regulation of immuneresponses. This embodiment can find particular use for cancer therapywhere regulatory T cells suppress immunotherapy and interventions.Regulatory T cells express MHC class II and can be targeted viaPADRE-dendrimer or other CD4 epitope-dendrimer. Another example isCTLA-4 on CD4 T cells that transmits an inhibitory signal to T cells. Inan embodiment wherein siRNA specific for CTLA-4 is complexed with aPADRE-dendrimer, the complex targets MHC class II expressing-cellsincluding CD4 T cells and silences the CTLA-4 expression. The lower theexpression of CTLA-4, the higher the immune responses against thepathogen or cancer, and when delivered into PAPCs, it prevents orreduces expression of molecules that inhibit immune responses (toenhance immune responses against pathogens, cancers, or when host isvaccinated) or enhances immune responses including B7.1, LFA-3, ICAM-1(inducer of signal 2 needed for activation of T cells) for the therapyof autoimmune diseases such as Psoriasis.

In a typical embodiment, a composition described herein includes ansiRNA specific to co-inhibitory and co-stimulatory molecules and theirputative co-stimulatory receptor(s)) (e.g., Foxp3, CD28 CTLA-4).Sequence-specific siRNAs bind to a target nucleic acid molecule,inhibiting the expression thereof. siRNAs are effective in the treatmentof abnormal cells, abnormal cell growth and tumors, including thosetumors caused by infectious disease agents. Compositions for delivery ofsiRNA and methods of treatment thereof are provided.

Methods of constructing and using ribozymes, siRNA and antisensemolecules are known in the art (e.g., Isaka Y., Curr Opin Mol Ther vol.9:132-136, 2007; Sioud M. and Iversen P. O., Curr Drug Targets vol.6:647-653, 2005; Ribozymes and siRNA Protocols (Methods in MolecularBiology) by Mouldy Sioud, 2^(nd) ed., 2004, Humana Press, New York,N.Y.). An “antisense” nucleic acid can include a nucleotide sequencewhich is complementary to a “sense” nucleic acid encoding a protein,e.g., complementary to the coding strand of a double-stranded cDNAmolecule or complementary to an mRNA sequence. The antisense nucleicacid can be complementary to an entire coding strand of a gene ofinterest, or to only a portion thereof. In another embodiment, theantisense nucleic acid molecule is antisense to a “noncoding region” ofthe coding strand of a nucleotide sequence encoding a gene of interest(e.g., the 5′ and 3′ untranslated regions). Anti-sense agents caninclude, for example, from about 8 to about 80 nucleobases (i.e. fromabout 8 to about 80 nucleotides), e.g., about 8 to about 50 nucleobases,or about 12 to about 30 nucleobases. Antisense compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides whichhybridize to the target nucleic acid and modulate its expression.Anti-sense compounds can include a stretch of at least eight consecutivenucleobases that are complementary to a sequence in the target gene(i.e., gene of interest). An oligonucleotide need not be 100%complementary to its target nucleic acid sequence to be specificallyhybridizable. An oligonucleotide is specifically hybridizable whenbinding of the oligonucleotide to the target interferes with the normalfunction of the target molecule to cause a loss of utility, and there isa sufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment or, in the case of invitro assays, under conditions in which the assays are conducted.

RNA Interference (RNAi) is a remarkably efficient process wherebydouble-stranded RNA (dsRNA, also referred to herein as siRNAs, for smallinterfering RNAs, or ds siRNAs, for double-stranded small interferingRNAs) induces the sequence-specific degradation of homologous mRNA inanimals and plant cells (Hutvagner and Zamore, Curr. Opin. Genet. Dev.,12:225-232 (2002); Sharp, Genes Dev., 15:485-490 (2001)). In mammaliancells, RNAi can be triggered by duplexes of small interfering RNA(siRNA) (Chiu et al., Mol. Cell., 10:549-561 (2002); Elbashir et al.,Nature, 411:494-498 (2001)), or by micro-RNAs (miRNA), functionalsmall-hairpin RNA (shRNA), or other dsRNAs which are expressed in vivousing DNA templates with RNA polymerase III promoters (Zeng et al., Mol.Cell, 9:1327-1333 (2002); Paddison et al., Genes Dev., 16:948-958(2002); Lee et al., Nature BiotechnoL, 20:500-505 (2002); Paul et al.,Nature BiotechnoL, 20:505-508 (2002); Tuschl, T., Nature Biotechnol.,20:440-448 (2002); Yu et al., Proc. Natl. Acad. Sci USA, 99(9):6047-6052(2002); McManus et al., RNA, 8:842-850 (2002); Sui et al., Proc. Natl.Acad. Sci. USA, 99(6):5515-5520 (2002)).

The dsRNA molecules typically include 16-30, e.g., 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand,wherein one of the strands is substantially identical, e.g., at least80% (or more, e.g., 85%, 90%, 95%, or 100%) identical, e.g., having 3,2, 1, or 0 mismatched nucleotide(s), to a target region in the mRNA, andthe other strand is identical or substantially identical to the firststrand. Each strand can also have one or more overhanging (i.e.,non-complementary) nucleotides, e.g., one, two, three, four or moreoverhanging nucleotides, e.g., dTdTdT.

The dsRNA molecules can be chemically synthesized, or can be transcribedin vitro from a DNA template, or in vivo from, e.g., shRNA. The dsRNAmolecules can be designed using any method known in the art; a number ofalgorithms are known in the art, see, e.g., Tuschl et al., Genes Dev13(24):3191-7 (1999), and many are available on the internet.

Negative control siRNAs typically have the same nucleotide compositionas the selected siRNA, but without significant sequence complementarityto the appropriate genome. Such negative controls can be designed byrandomly scrambling the nucleotide sequence of the selected siRNA; ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence. In some embodiments, siRNA can be producedusing modified nucleotides (e.g., 2F-RNA) to make the siRNA resistant tonucleases.

Methods and Kits for Generating Antibodies

Compositions, kits and methods for generating antibodies that can beadministered to a subject for therapeutic or prophylactic purposes aredescribed herein. Current methods of DNA delivery into cells areinefficient, complex, and induce poor immune responses. The compositionsand methods described herein, however, result in a strong antibodyresponse that demonstrates rapid and high expression of an antigen ofinterest. The dendrimer/T helper peptide conjugates (e.g.,PADRE-dendrimers) described herein can be complexed with (conjugated to)peptides or polypeptides or a nucleic acid (e.g., DNA) in a method ofgenerating antibodies. Binding poly(I:C) to dendrimers is of particularuse for the production of monoclonal antibodies because it reduces thefrequency of and intervals between injections. In the experimentsdescribed below, delivery and expression of GFP in the cornea and skinas well as strong humoral responses were shown after a single injectionof PADRE-dendrimer complexed with DNA. 50% of current drugs targetmembrane proteins that are the most difficult to purify. Thecompositions, kits and methods described herein provide tools to targetsuch difficult-to-purify proteins without a need for purifying them.

Polyclonal antibodies are heterogeneous populations of antibodymolecules that are contained in the sera of the immunized animals.Antibodies that can be produced using the compositions, kits and methodsdescribed herein therefore include polyclonal antibodies and, inaddition, monoclonal antibodies, single chain antibodies, Fab fragments,F(ab′)₂ fragments, and molecules produced using a Fab expressionlibrary. Monoclonal antibodies, which are homogeneous populations ofantibodies to a particular antigen, can be prepared using thedendrimer/T helper epitope conjugates described herein and standardhybridoma technology (see, for example, Kohler et al., Nature 256:495,1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur.J. Immunol. 6:292, 1976; Hammerling et al., “Monoclonal Antibodies and TCell Hybridomas,” Elsevier, N.Y., 1981; Ausubel et al., supra). Inparticular, monoclonal antibodies can be obtained by any technique thatprovides for the production of antibody molecules by continuous celllines in culture such as described in Kohler et al., Nature 256:495,1975, and U.S. Pat. No. 4,376,110; the human B-cell hybridoma technique(Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Natl.Acad. Sci. USA 80:2026, 1983), and the EBV-hybridoma technique (Cole etal., “Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, Inc., pp.77-96, 1983). Such antibodies can be of any immunoglobulin classincluding IgG, IgM, IgE, IgA, IgD and any subclass thereof. A hybridomaproducing a mAb as described herein may be cultivated in vitro or invivo. The ability to produce high titers of mAbs in vivo makes thecompositions, kits and methods described herein particularly useful formAb production.

The compositions, kits and methods described herein in which adendrimer/T helper peptide (e.g., PADRE-dendrimers) is complexed with(conjugated or bound to) a nucleic acid encoding a protein or antigennegate the need for the purification of protein or antigen since thenucleic acid (e.g., plasmid DNA or mRNA) encoding the antigen or proteinoffer the advantages of i) elimination of tedious and/or costly and/ortimely steps of protein purification, ii) the expression of the nativeform of the protein in vivo by cell machinery of the host which negatesthe challenge of a non-native form of the protein that results fromconventional protein purifications, and ii) an ideal method forgeneration of therapeutic monoclonal antibodies where the natural/nativeform of the protein or antigen is the target.

Human antibodies against a particular antigen can be made by adaptingknown techniques for producing human antibodies in animals such as mice.See, e.g., Fishwild, D. M. et al., Nature Biotechnology 14 (1996):845-851; Heijnen, I. et al., Journal of Clinical Investigation 97(1996): 331-338; Lonberg, N. et al., Nature 368 (1994): 856-859;Morrison, S. L., Nature 368 (1994): 812-813; Neuberger, M., NatureBiotechnology 14 (1996): 826; and U.S. Pat. Nos. 5,545,806; 5,569,825;5,877,397; 5,939,598; 6,075,181; 6,091,001; 6,114,598; and 6,130,314.Humanoid or humanized antibodies against a particular antigen can bemade from non-human antibodies by adapting known methods such as thosedescribed in U.S. Pat. Nos. 5,530,101, 5,585,089, 5,693,761, and5,693,762.

Once produced, polyclonal or monoclonal antibodies can be tested forspecific antigen recognition by Western blot, immunoprecipitationanalysis by standard methods or other suitable methods, for example, asdescribed in Ausubel et al., supra. Antisera can be raised by injectionsin a series, preferably including at least three booster injections.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

A typical method of delivering an antigen to a mammal and inducingproduction of monoclonal antibodies against the antigen in the mammalfor the purpose of obtaining monoclonal antibodies includesadministering to the mammal a composition including at least onedendrimer having conjugated thereto at least one T helper peptide and apeptide or polypeptide antigen or a nucleic acid encoding the antigenwherein the at least one T helper peptide and the nucleic acid orpeptide or polypeptide antigen are conjugated to the exterior surface ofthe at least one dendrimer such that the at least one T helper peptidespecifically binds to PAPCs and the combination of the at least one Thelper peptide, at least one dendrimer, and the nucleic acid or peptideor polypeptide antigen are able to induce an immune response against theantigen. In this method, the composition is administered in an amounteffective to induce MHC class II mediated activation of helper T cells,resulting in production of monoclonal antibodies against the antigen. Ina method of producing antibodies in mice, after the mice are immunizedwith antigen as described above, antibodies are harvested from one ormore mice, and are screened for high titer. A mouse with high titer isselected, and the spleen from this mouse is removed. Fusion with myelomais then performed, and screening for the best binding clone isperformed.

Also described herein are kits for generating antibodies (e.g.,monoclonal antibodies) to an antigen that eliminate the need for proteinpurification. A typical kit includes a container that includes aplurality of dendrimer/T helper peptide complexes (conjugates) asdescribed herein (e.g., PADRE-derivatized dendrimers, dendrimersconjugated to influenza HA, etc.), and a physiological buffer, typicallywith a pH of 7.4. In one example of a buffer or medium, the buffer ormedium includes Eagle's Minimal Essential Medium, buffered with HEPESand sodium bicarbonate, and supplemented with hypoxanthine, thymidine,sodium pyruvate, L-glutamine, and less than 10% serum bovine albumin orindividual serum proteins including insulin and/or teansferrin with 100mg/L CaCl2 where the endotoxin level is less than 1.0 EU/mL. In thisembodiment, a user of the kit dilutes at least one nucleic acid (e.g.,DNA plasmid) encoding one antigen with the buffer at 100-200 μg/ml, andwhile shaking gently, adds the composition (T helper-dendrimer) to thediluted plasmid DNA. In a typical embodiment, a ratio of 10:1 of Thelper-dendrimer to plasmid DNA is used (N:P), which is approximately 7times (weight) of composition to one time (weight) of DNA plasmid(s). Inone embodiment, the following conditions are followed. After 10 minutesincubation at room temperature, 100 ul of the mixture containing 10-20μg of the plasmid(s)-DNA/composition is subcutaneously injected in mice.A similar booster in 14 days is followed by standard methods of primaryscreening, fusion and final screenings for monoclonal antibodies. Inaddition to nucleic acids, the compositions and kits described hereinmay be conjugated to proteins or antigens. In such an embodiment,typically the same ratio of 10:1 of the composition to protein resultsin the complex formation. The instructions for use included in a kit asdescribed herein describes the protocol of making proper ratios,buffers, and optimization and troubleshooting when needed. Complexationof plasmid DNA or protein/antigen with the PADRE-dendrimer conjugatesdescribed herein is generally done by mixing the two components inaqueous solution buffered at physiological pH with a physiologicalbuffer including PBS. Typical N/P (amine to phosphate) ratios are 10:1.Gel electrophoresis or other suitable assay can be used to demonstratecomplete complexation of the DNA to the PADRE-dendrimer.

A kit as described herein can be used with any vector or plasmidencoding an antigen of interest. Instructional materials for preparationand use of the dendrimer/T helper eptiope complexes (conjugates)described herein are generally included. While the instructionalmaterials typically include written or printed materials, they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is encompassed by the kits and methodsherein. Such media include, but are not limited to electronic storagemedia (e.g., magnetic discs, tapes, cartridges, chips), optical media(e.g., CD ROM), and the like. Such media may include addresses tointernet sites that provide such instructional materials.

Compositions and Methods for Delivering a Nucleic Acid to a Cell

In the experiments described herein, delivery of a gene encoding GFP wasspecifically delivered to MHC Class II cells (cells expressing MHC ClassII) and expression of the gene was observed. Thus, the compositions andmethods described herein may find use in any gene therapy application. Acomposition for delivering a nucleic acid to a cell typically includesat least one positively-charged highly branched polymeric dendrimerhaving conjugated thereto at least one T helper peptide and at least onenucleic acid encoding a peptide or protein, wherein the at least one Thelper peptide and the nucleic acid are conjugated to the exteriorsurface of the at least one positively-charged highly branched polymericdendrimer such that the at least one T helper peptide specifically bindsto the cell, and the combination of the at least one T helper peptide,at least one positively-charged highly branched polymeric dendrimer, andthe nucleic acid are internalized by the cell. A method of delivering anucleic acid to a cell typically includes contacting the cell with acomposition including at least one positively-charged highly branchedpolymeric dendrimer having conjugated thereto at least one T helperpeptide and at least one nucleic acid encoding a peptide or protein,wherein the at least one T helper peptide and the nucleic acid areconjugated to the exterior surface of the at least onepositively-charged highly branched polymeric dendrimer such that the atleast one T helper peptide specifically binds to the cell, and thecombination of the at least one T helper peptide, at least onepositively-charged highly branched polymeric dendrimer, and the nucleicacid are internalized by the cell. In a typical embodiment, the peptideor protein is expressed within the cell.

Administration of Compositions

The vaccines and compositions described herein may be administered tomammals (e.g., dog, cat, pig, horse, rodent, non-human primate, human)in any suitable formulation. For example, a composition including aPADRE-dendrimer conjugated to siRNA or a vaccine including aPADRE-dendrimer complexed to a nucleic acid, or peptide or polypeptideantigen may be formulated in pharmaceutically acceptable carriers ordiluents such as physiological saline or a buffered salt solution.Suitable carriers and diluents can be selected on the basis of mode androute of administration and standard pharmaceutical practice. Adescription of exemplary pharmaceutically acceptable carriers anddiluents, as well as pharmaceutical formulations, can be found inRemington's Pharmaceutical Sciences, a standard text in this field, andin USP/NF. Other substances may be added to the compositions tostabilize and/or preserve the compositions.

The compositions and vaccines described herein may be administered tomammals by any conventional technique. Typically, such administrationwill be parenteral (e.g., intravenous, subcutaneous, intratumoral,intramuscular, intraperitoneal, or intrathecal introduction). Thecompositions may also be administered directly to a target site. Thecompositions may be administered in a single bolus, multiple injections,or by continuous infusion (e.g., intravenously, by peritoneal dialysis,pump infusion). For parenteral administration, the compositions arepreferably formulated in a sterilized pyrogen-free form. In therapeuticapplications, the compositions and vaccines described herein areadministered to an individual already suffering from cancer, or infectedwith the pathogen (e.g., virus) of interest. In prophylacticapplications, the compositions and vaccines described herein areadministered to an individual at risk of developing (e.g., geneticallypredisposed to, or environmentally exposed to) cancer or an infectiousdisease (i.e., infected with a pathogen (e.g., virus) of interest).

In therapeutic applications, the compositions and vaccines describedherein are administered to an individual already suffering from cancer,or infected with the pathogen (e.g., virus) of interest. In prophylacticapplications, the compositions and vaccines described herein areadministered to an individual at risk of developing (e.g., geneticallypredisposed to, or environmentally exposed to) cancer or an infectiousdisease (i.e., infected with a pathogen (e.g., virus) of interest).

Effective Doses

The vaccines and compositions described herein are preferablyadministered to a mammal (e.g., dog, cat, pig, horse, rodent, non-humanprimate, human) in an effective amount, that is, an amount capable ofproducing a desirable result in a treated mammal (e.g., prevention orelimination of cancer in a mammal, protection against infectiousdisease(s), etc.). Such a therapeutically effective amount can bedetermined as described below.

Toxicity and therapeutic efficacy of the vaccines and compositionsdescribed herein can be determined by standard pharmaceuticalprocedures, using either cells in culture or experimental animals todetermine the LD₅₀ (the dose lethal to 50% of the population). The doseratio between toxic and therapeutic effects is the therapeutic index andit can be expressed as the ratio LD₅₀/ED₅₀. Those compositions thatexhibit large therapeutic indices are preferred. While those thatexhibit toxic side effects may be used, care should be taken to design adelivery system that minimizes the potential damage of such sideeffects. The dosage of preferred compositions lies preferably within arange that includes an ED₅₀ with little or no toxicity. The dosage mayvary within this range depending upon the dosage form employed and theroute of administration utilized.

Therapeutically effective amounts of the compositions and vaccinesdescribed herein generally range for the initial immunization (that isfor therapeutic or prophylactic administration) from about 1 μg to about25,000 μg (e.g., 1, 100, 500, 2000, 2500, 10,000, 15,000, 25,000 μg) ofa complex of T helper epitope/dendrimer conjugated to antigen or boundto a nucleic acid encoding the antigen for a 70 kg patient (e.g., 0.14μg to 357 μg of plasmid(s) DNA or protein and 0.86 μg to 2142.85 μg ofthe T-helper-dendrimer), followed by boosting dosages of from about 1 μgto about 2500 μg of the complex (vaccine) pursuant to a boosting regimenover weeks to months depending upon the patient's response and conditionby measuring specific CTL activity and/or antibody responses in thepatient's blood. In one embodiment, 15 daily administrations ofdendrimer in doses >133-fold greater then the above doses may beadministered to a mammal with no toxicity (see Abhay Singh Chauhan et.al. 2009 Proc. R. Soc. A, 466, pp 1535-1550. 2009).

For treating a subject currently suffering from cancer or an infectiousdisease and/or who has just been diagnosed with cancer or an infectiousdisease, administration preferably begins at the first sign of diseaseor the detection or surgical removal of tumors or shortly afterdiagnosis in the case of acute infection. This is followed by boostingdoses until at least symptoms are substantially abated and for a periodthereafter. In chronic infection, loading doses followed by boostingdoses may be required. For prophylactic use, administration may begin assoon as an individual becomes aware of a predisposition to cancer, orprior to an expected exposure to an infectious disease.

As is well known in the medical and veterinary arts, dosage for any onesubject depends on many factors, including the subject's size, bodysurface area, age, the particular composition to be administered, timeand route of administration, general health, and other drugs beingadministered concurrently. Dendrimers such as PAMAM have been tested inpreclinical studies as well as in clinical trials. Recently, the FDAgranted a “fast track” status to VivaGel® (Starpharma, Melbourne,Australia), already in phase III clinical trials. Therapeutic use ofdendrimers in the cornea is known, and dendrimers have been used incorneal gene delivery. Examples of using dendrimers in cornealapplications include the therapy of corneal neovascularization,photodynamic therapy, and tissue-engineering as a corneal equivalent.Poly(I:C) has been administered to humans for more than 40 years as a“natural” inducer of Interferon. Several recent clinical trials haveexamined different doses and routes of administration for safety andenhanced immunogenicity; general safety and enhanced immunogenicity havebeen repeatedly reported and established.

Examples

The present invention is further illustrated by the following specificexamples. The examples are provided for illustration only and should notbe construed as limiting the scope of the invention in any way.

Example 1—an Adjuvanted/Targeted Nanoparticle-Based Platform for GeneticVaccination Therapy of Established Melanoma Tumors

Genetic vaccination using naked DNA is used to produce antigens in theirnatural forms. However, the low in vivo transfection efficacy, a lack ofeffective delivery and the poor specificity of current geneticvaccination approaches strongly limit their efficacy. To overcome theselimitations, a novel and flexible platform for antitumor DNA vaccinationthat 1) allows the specific and efficient transfection of PAPCs in vivo,2) provides “danger signals” that result in maturation of autologousPAPCs and hence robust immune responses and, 3) activates helper T cellsthat further boost the generated immune responses was developed and isdescribed herein.

The novel dendrimer-based nanoparticles described herein are typicallyprepared by the conjugation of two reactants: a fifth-generation,amino-terminated, PAMAM dendrimer, and a targeting/immune-enhancingpeptide, or universal T helper Epitope (PADRE). The data described belowshowed this platform to i) achieve an objective anti-tumor effect with areduction in tumor size of 50% of established and highly aggressiveB16/LU8 melanoma tumors in C57BL mice, ii) induce a robust immuneresponse against the product of the gene used in vaccination, and ii)increase transfection efficiency in both mouse and human APCs by 2- to3-fold. Moreover, in vivo experiments using GFP-encoding plasmidconjugated to PADRE-dendrimer showed that GFP is produced in thedraining lymph nodes.

Materials and Methods

PADRE-derivatized PAMAM dendrimer was generated as described above withthe following modifications. The PADRE-dendrimer/DNA or siRNA complexwas generated by incubation at room temperature for 10 minutes at aproper N/P ratio. Such complexes were added to primary PBMC orsplenocytes for in vitro studies or injected subcutaneously forvaccination purposes. FIG. 1 shows PADRE decoration of (conjugation to)fifth-generation PAMAM Dendrimer.

To maintain the highly positively-charged surface for binding ofmultiple nucleic acids, one dendrimer molecule typically has two PADREpeptides conjugated to its surface so that it will still keep itspositive net charge. Addition of PADRE to the dendrimers results inspecific targeting of APCs, and strong CD4 help. The PADRE-derivatizedPAMAM dendrimers not only escort plasmid-encoding antigens but alsostimulate innate and adaptive immunity and act as a “danger signal.”

Endotoxin-free MaxiPrep reagents were used to produce various plasmids(including pEGFP-C1, pMAX, GFP, TRP-2, P2, PCARD, and OVA in PCDNA3.1).Flourochrome-linked Immunosorbent Assays (FLISA), and Immuno FlorescenceAssays (IFA) were performed by standard methods. Briefly, transfectedcos-7 cells were plated (0.02×10⁶ per well in a 96-well plate), cellswere fixed and permeabilized. To measure mounted humoral responses,diluted sera were added to the wells and a secondary anti-mouseIgG-tagged with IRDye 800CW was used to measure antibody responses.

To prepare the Dendrimer/DNA complex, 1 μg/μL of prepared DNA inendotoxin-free PBS was complexed with dendrimer or dendrimer-PADRE invarious charge ratios. After a 10 min incubation at room temperature,the complexes were added to cell culture or injected subcutaneously,intradermaly, or into the corneal stroma cavity.

For the vaccination of mice bearing B16-LU8 melanoma tumors, femaleC57BL mice in groups of five were implanted with (0.02×10⁶) B16-F10cells, subcutaneously. Different groups received i) no treatment, ii)PCDNA3.1 (vector control) alone, iii) TRP-2 complexed with dendrimeralone, or iv) TRP-2 complexed with PADRE-dendrimer, on day two and tenpost-tumor implantation. Tumor measurements were performed twice a week.

Results

The prepared PADRE-dendrimers were characterized. The peptide-dendrimerconjugate was made by simple amide coupling between the —COOH terminusof the peptide and the dendrimer amine groups. A 2:1 peptide/dendrimerchallenge ratio was used in the reaction, seeking attachment of just afew peptides per dendrimer, in order to keep most of the free aminegroups to develop large positive charges on the dendrimer. The productwas purified by dialysis against pure water for at least 24 h and thendried under vacuum. The collected product, a clear oil, wascharacterized by 1H NMR, UV-Vis and MALDI-TOF mass spectroscopy. NMRshows large peaks corresponding to the dendrimer protons and a small setof peaks for the peptide protons. The MALDI-TOF mass spectrum of thePADRE-dendrimer conjugate shows a peak at a m/z ratio ca. 3,000 unitshigher than the peak observed for the dendrimer on its own. The excessmass corresponds to approximately 2 peptide epitopes (FIG. 17).

The data established that an average of two PADRE are present on eachdendrimer (FIG. 17). In vitro delivery of multiple nucleic acids intoautologous APCs was shown. In vitro multinucleotidedelivery/transfection of human primary peripheral mononuclear cells wasbest achieved in the charge ratios of 1:5 and 1:10. FIG. 2 shows siRNAdelivery (˜%86) via PADRE-dendrimers into purified human B cells whereAlexa Fluor-tagged siRNA complexed with (conjugated to) PADRE-dendrimerwas incubated with B cells for 4 hours. Cells were stained withCD19/FITC and the red channel (PE) represents cells with the siRNA/AlexaFluor.man

Referring to FIG. 3, in vivo DNA delivery of PADRE-dendrimers was shown.Plasmids encoding GFP or TRP-2 were injected alone or complexed withPADRE-dendrimer, or dendrimer (i.e., dendrimer not complexed withPADRE). The images show the expression of GFP in skin (left) and cornea(right) 24 and 16 hours post-injection. Effective expression of GFP isdemonstrated in both skin and cornea 24 and 16 hours post-injection ofPADRE-dendrimer complexes. Targeting of the lymph nodes in vivo wasdemonstrated. Eight days after PADRE-dendrimer/GFP-plasmid complexeswere injected subcutaneously (5 μg total plasmid), the adjacent lymphnode was removed and compared with lymph nodes of a mouse injected withGFP-DNA alone. Fluorescent microscope images were taken on meshed lymphnodes on day eight post-immunization. Expression of antigen in the lymphnode adjacent to the injection site was seen, but expression of antigenin a control lymph node was not seen.

Targeting of the lymph nodes in vivo was demonstrated. Eight days afterPADRE-dendrimer/GFP-plasmid complexes were injected subcutaneously (5 μgtotal plasmid), the adjacent lymph node was removed and compared withlymph nodes of a mouse injected with GFP-DNA alone. Fluorescentmicroscope images were taken on meshed lymph nodes on day eightpost-immunization. Expression of antigen in the lymph node adjacent tothe injection site was seen, but expression of antigen in a controllymph node was not seen.

As shown in FIG. 4, specific immune responses were mounted afteradministration of PADRE-dendrimer/plasmid complexes. Strong humoralresponses were observed upon one (GFP) or two immunizations (OVA) withcomplexes of plasmid/PADRE-dendrimer.

The data shown in FIG. 5 shows PADRE-dendrimer therapy of establishedtumors. B16 melanoma is known to be an aggressive mouse tumor model.Mice implanted with B16 melanoma cells (top) or TSA (bottom) werevaccinated on day two or three post-tumor implantation followed withbooster immunizations after a week. Follow up of tumor measurementsclearly demonstrated that administration of the PADRE-dendrimersresulted in a protective immune response, in particular, in C57BL (lab)which has higher affinity for PADRE binding. C57BL also respondsstronger to PADRE via induction of T helper cells.

Referring to FIG. 5, the results demonstrated an objective anti-tumoreffect with a reduction in tumor size of 50% of the highly aggressiveand established B16/LU8 tumor. This aggressive model was chosenintentionally to show the potency of the platform as everything elsefails. The delay in tumor growth and reduction in tumor size isunprecedented. Indeed, on day 22 post-tumor implantation, no or nopalpable tumors were detected in the test group (i.e., those animalsreceiving a vaccine as described herein) which was significantlydifferent from all control groups. The amount of DNA used in thisvaccination (vaccine dose) was much lower than what is normally used (20μg and a total of 2 immunizations). In FIG. 5, the lower figure is anegative control using Balb/c mice where PADRE does not bind properly, asimilar experiment in a Balb/c TSA tumor model shows no efficacy. Thisclearly shows the specificity of the delivery system via MHC class II.

These data clearly demonstrate that the targeted adjuvanted nanopatricleplatform described herein results in gene delivery, robust expression ofthe encoded antigen, antigen presentation, and induction of protectiveimmune responses. Thus, the PADRE-dendrimer nanoparticles describedherein are a novel and powerful adjuvanted/targeted delivery tool andplatform for: i) protein-free generation of (monoclonal) antibodies, ii)immunological treatment/prevention of malignancies and infectiousdiseases with deceptive imprinting, and iii) delivery of siRNA for manyimmune-based therapeutic interventions.

Example 2—In Vitro Targeted Delivery and Transfection Efficiency

Referring to FIG. 6, in vitro targeted delivery of PBMCs results in 77%B cell transfection efficiency. Human PBMC from healthy donors wereobtained. PBMCs were cultured at 6 million cells per ml of RPMI mediawith 10% fetal bovine serum. The plasmid encoding for GFP at 5 μg wasdiluted in 100 ul of a physiological buffer, PBS, and 5 μg ofPADRE-dendrimer in 50 ul PBS was added to DNA while shaking. After 10minutes incubation at room temperature, the mixture/complex of the GFPplasmid and PADRE-dendrimer was then added to PBMC. Twenty-four hourspost incubation at 37° C./5% CO₂ incubator, PBMCs were stained with CD19PE and cells were analyzed by flow cytometry. The expression of GFP wasobserved in 43% of total PBMC while when gated on B cells 77% of B cellsexpressed GFP. Control groups, PBMC incubated with same ratios ofdendrimer and GFP plasmid showed about 11% and 7% GFP expression intotal PBMC or B cells. No major viability change was observed whencompared with PBMC with only media. This is a representative experimentof several. These experiments demonstrate i) the delivery of GFP plasmidinto PBMC and in particular to MHC class II expressing cells (B cells),and ii) the expression of the GFP by PBMC and in particular by B cells.

Example 3—Delivery of Peptides/Proteins into Mouse DCs In Vivo and HumanB Cells In Vitro

PDD/Albumin-FITC was delivered into purified human B cells (FIG. 7).Referring to FIG. 8, this Figure shows PADRE-dendrimer targeting of andefficacy in mouse DCs in vivo and a timeline for injection and lymphnode analysis. The results of this experiment show that i) (FIG. 7)Albumin-FITC, a protein, mixed with PADRE-dendrimer was delivered inhuman B cells in less than two hours, ii) (FIG. 8) in day 5 postsubcutaneous injection, PADRE, an epitope, conjugated to dendrimer wasdelivered into lymph node's B cells and DCs in vivo (the PADRE-dendrimerwas complexed to GFP-plasmid to visualize the delivery of the complex tolymph node/B cells/DCs.), iii) (FIG. 9) in day 5 post subcutaneousinjection, HA helper epitope of influenza, an epitope, conjugated todendrimer was delivered into lymph node's DCs in vivo (thePADRE-dendrimer was complexed to GFP-plasmid to visualize the deliveryof the complex to lymph node DCs). These data were representative ofseveral experiments and in some the lymph nodes were removed on day 3post subcutaneous injection of PADRE-dendrimer or HA-dendrimer eachcomplexed with GFP plasmid. These results establish examples of thedelivery to APCs including B cells and DC of a protein conjugated withFITC via FITC visualization of FITC as well as the delivery of twopeptides, PADRE and HA helper epitopes conjugated to dendrimer where GFPplasmid was complexed with the peptide-dendrimer to facilitatevisualization and analysis of the complex (peptide-linked to dendrimercomplex with GFP-encoding plasmid) in the cells of lymph nodes. Specificin vitro and in vivo transfection of DCs was shown by in vivo flowcytometry data on targeting and expanding DCs in an adjacent lymph node,5-days post-injection of the nanoparticle (PADRE-dendrimer/GFP-encodingplasmid) vs. controls (78% vs. ˜7% GFP expression). ThePADRE-derivatized dendrimer (PDD) enhances delivery due to its assistedopsonized effect of PADRE which with high affinity binds to MHC class IIexpressed on APC. Similarly, HA-dendrimer (DRHA)/GFP-plasmid wasdelivered in vivo in the neighboring lymph nodes, when injectedsubcutaneously (FIG. 9). Note that in mice, PADRE binds the MHC class IIof IAb (C57BL mice) (FIG. 8) while selected HA epitope binds the MHCclass II of IAd (Balb/c mice) (FIG. 9). The feasibility of in vivodelivery in two different mice strains with two different epitopes withsimilar results have been shown. The APC-targeted delivery resulting inthe expression of GFP by PADRE-dendrimer/GFP-plasmid into human PBMCs(FIG. 6), purified human B cells (FIG. 6), and in splenocytes of C57BLmice, and the delivery of PADRE-dendrimer/dsRNA into human B cells (FIG.10) and of monkey PBMC (FIG. 11) are additional in vitro evidence of thedelivery of peptide to PAPCs by the compositions described herein.Because use of two different targeting peptides, whose unique feature isto bind to the MHC class II, works as shown in the experiments describedherein, the vaccines, methods and compositions described hereinencompass all MHC class II binding peptides. Referring to FIG. 9,dendrimer conjugated to influenza HA helper epitope (HDD) was alsoprepared. HDD may be used in balb/c mice. When injected into mice, HDDtargets DCs in the adjacent lymph node.

Example 4—PADRE-Dendrimer Delivery of siRNA into Human B Cells andNon-Human Primate PBMCs and PADRE-Dendrimer Delivery of Plasmid intoNon-Human Primate PBMCs

Referring to FIG. 10, PADRE-dendrimers complexed to dssiRNA (a controlsiRNA) exhibited targeted delivery in vitro. 0.1 μg of dsRNA was dilutedin 100 ul of PBS and 0.7 μg of the PADRE-dendrimer in 20 ul was added todsRNA-Alexa Fluor tagged while shaking. The complex after a 10 minuteincubation at room temperature was added to one million purified B cells(in RPMI plus 10% fetal bovine serum) in wells of a 24-well plate. Aboutan hour post incubation at 37° C./5% CO₂ incubator, cells were washedand placed back in the wells (in 1 ml of fresh RPMI plus 10% fetalbovine serum) and were analyzed under fluorescent microscope in redchannel. The overlay image of cells under bright field and red channeldemonstrates the uptake of Alexa Fluor tagged dsRNA by human B cells(FIG. 10). Cells were incubated overnight at 37° C./5% CO₂ incubatorwhen they were stained with CD19 (a B-cells marker) and analysed by flowcytometry (FIG. 2). As shown in the FIG. 2, >80% of the B cells werepositive for Alexa Fluor (tagged to dsRNA) versus about 6% for thecontrol, dendrimer/dsRNA-Alexa Fluor. These results clearly demonstratethe robust delivery of nucleic acids to PAPC by PADRE-dendrimer.

PBMCs one sample from baboon (papio hamadryas), and two differentsamples from cynomolgus monkeys (macaca fascicularis) were tested.Fluorescent microscope images shown in FIG. 11 are representative, takentwo hours post-addition of PADRE-dendrimer or dendrimer, each complexedwith siRNA/Alexa Fluor. Similarly, PADRE-dendrimer or dendrimercomplexed with GFP-plasmid were added to the PBMCs and were analyzed 24hours after incubation (FIG. 12). The results show that, in less than 2hours, PADRE-dendrimer delivers nucleic acids into the monkeys' PBMCs,while dendrimer shows only a modest delivery. These results stronglysuggest that PADRE-dendrimer works on non-human primates.

Example 5—Comparison of PADRE-Dendrimer Nanoparticles and theIN-CELL-ART™ Platform

The PADRE-dendrimers described herein provide specific targeting ofPAPCs in contrast to IN-CELL-ART's non-specific delivery to all cells.The IN-CELL-ART™ platform includes a 704 polymer that delivers DNA tocells via electroporation with no specific built-in adjuvant activity orany ligand for binding APCs. Currently, in vivo electroporation is knownas the best non-viral genetic immunization method. The PADRE-dendrimersdescribed herein provide induction of helper T cells that is notprovided by the IN-CELL-ART's platform. In the experiments describedabove, the PADRE-dendrimers were shown to be efficacious in atherapeutic tumor model while IN-CELL-ART's has not been shown to beefficacious in an in vivo tumor model. In the experiments describedherein, as a robust control, PADRE-dendrimers were compared with in vivoelectroporation (FIG. 13). In contrast, IN-CELL-ART has only shown acomparison with naked DNA. A single immunization of the PADRE-dendrimersdescribed herein was compared with in vivo electroporation for mountinghumoral responses. To perform this experiment, PADRE-dendrimers weremixed with 5 μg of the plasmid at room temperature, and 10 minuteslater, the mixture was injected into mice subcutaneously. Mice immunizedwith PADRE-dendrimers conjugated to OVA-encoding plasmid and controlmice were challenged with 50k B16F10-expressing OVA on day 28 post asingle vaccination. All mice that received PADRE-dendrimers conjugatedto plasmid were protected on day 25 post-tumor implantation versus 40%of those receiving plasmid via in vitro electroporation and 0% in thecontrol group that received DNA only. As shown in FIG. 13, the resultsshow that a single DNA vaccination with PADRE-dendrimer/plasmid(DRP-ova) is superior to in vivo electroporation (EP) delivery ofplasmid (EP-ova) for induction of an anti-ova antibody humoral response.

Example 6—PADRE-Dendrimers Induce Strong Humoral Responses in Mice

As shown in FIGS. 14-16, PADRE-dendrimers complexed with a plasmidelicited strong humoral responses in mice.

Example 7—Eradication of B16/OVA Tumors in a Prophylactic Setting byPADRE-Dendrimer/OVA Plasmid Vaccine

Female C57BL mice, 6 weeks-old, in groups of five per cage received i)nothing, ii) two immunizations of 20 μg OVA-plasmid via “in vivoelectroporation” using Derma Vax electroporator, or iii) twoimmunization with PADRE-dendrimer/OVA-plasmid (20 μg each).Immunizations were performed 2 weeks apart. Ten days post immunizations,all mice received subcutaneous injections (implantation) of 50,000B16/OVA tumor cells in 100 μl PBS in the right flanks. Tumormeasurements were performed twice a week and weekly data was plotted andis shown in FIG. 18. Two vaccinations with PADRE-dendrimer/20 μgOVA-plasmid resulted in complete eradication of B16/OVA tumors in allimmunized mice while 100% of no treatment and 60% of mice vaccinated via“in vivo electroporation” remained tumor-bearing. All tumor bearing micewith tumors larger than 15% of the body weight were sacrificed.

OTHER EMBODIMENTS

Any improvement may be made in part or all of the compositions, kits,and method steps. All references, including publications, patentapplications, and patents, cited herein are hereby incorporated byreference. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended to illuminate the invention anddoes not pose a limitation on the scope of the invention unlessotherwise claimed. For example, although the experiments describedherein involve eradication of B16 melanomas and induction of stronghumoral responses to GFP, OVA, PCARD, CCR5, vgPCR, muPAR, CathL, or p2antigens, the vaccines, compositions and methods described herein canfind use in a number of other therapeutic and prophylactic applications,including preventing or eradicating additional types of cancer, andinducing an immune response and thus immunity against any antigen ofinterest (e.g., antigens from infectious pathogens). In another example,the vaccines, compositions and methods described herein can be used todeliver a protein or peptide that is not an antigen to a cell. In thisexample, a typical composition for delivering a peptide or protein to acell includes at least one charged highly branched polymeric dendrimerhaving conjugated thereto at least one T helper peptide and at least onepeptide or protein, wherein the at least one T helper peptide and the atleast one peptide or protein conjugated to the exterior surface of theat least one charged highly branched polymeric dendrimer such that theat least one T helper peptide specifically binds to the cell. Anystatement herein as to the nature or benefits of the invention or of thepreferred embodiments is not intended to be limiting, and the appendedclaims should not be deemed to be limited by such statements. Moregenerally, no language in the specification should be construed asindicating any non-claimed element as being essential to the practice ofthe invention. This invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedelements in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontraindicated by context.

1. A conjugate comprising at least one charged highly branched polymericdendrimer having conjugated thereto at least one T helper peptide thatspecifically binds to a professional antigen presenting cell and atleast one nucleic acid molecule comprising a sequence encoding at leastone antigen.
 2. The conjugate of claim 1, wherein the at least one Thelper peptide is a Pan-DR epitope (PADRE).
 3. The conjugate of claim 1,wherein the at least one T helper peptide comprises the amino acidsequence of any of SEQ ID NOs:1 and 2-24.
 4. The conjugate of claim 1,wherein the nucleic acid molecule comprises an expression controlsequence operably linked to the sequence encoding the at least oneantigen.
 5. The conjugate of claim 1, wherein the at least one antigenis a cancer antigen or an antigen from an infectious pathogen.
 6. Theconjugate of claim 1, wherein the at least one T helper peptide isinfluenza HA.
 7. The conjugate of claim 1, wherein the at least onecharged highly branched polymeric dendrimer is a PAMAM dendrimer.
 8. Acomposition comprising the conjugate of claim 1, a pharmaceuticallyacceptable carrier and an oil and water emulsion.
 9. A conjugatecomprising at least one charged highly branched polymeric dendrimerhaving conjugated thereto at least one T helper peptide thatspecifically binds to a professional antigen presenting cell and atleast one antigen.
 10. The conjugate of claim 9, wherein the at leastone charged highly branched polymeric dendrimer has further conjugatedthereto a second peptide comprising an antigen that is different fromthe at least one antigen. 11.-24. (canceled)